Murine, chimeric, humanized or human anti-IL-6 antibodies

ABSTRACT

The present invention concerns compositions and methods of use of bispecific antibodies comprising at least one anti-TNF-α antibody or antigen-binding fragment thereof and at least one anti-IL-6 antibody or antigen-binding fragment thereof. Preferably, the bispecific antibody is in the form of a DNL® complex. The anti-TNF-α or anti-IL-6 antibodies may comprise specific CDR sequences disclosed herein. The compositions and methods are of use to treat autoimmune disease, immune system dysfunction or inflammatory disease, as disclosed herein.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/206,571, filed Jul. 11, 2016 (now abandoned), which was a divisionalof U.S. patent application Ser. No. 14/525,690 (now issued U.S. Pat. No.9,416,197), filed Oct. 28, 2014, which claimed the benefit under 35U.S.C. 119(e) of provisional U.S. Patent Application Ser. No.61/898,798, filed Nov. 1, 2013.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 27, 2014, isnamed IBC139US1_SL.txt and is 58,035 bytes in size.

FIELD OF THE INVENTION

The present invention relates to compositions and methods of use ofcomplexes comprising at least one anti-TNF-α antibody or antigen-bindingfragment thereof and at least one anti-IL-6 antibody or antigen-bindingfragment thereof. The complex may be a bispecific or multispecificantibody or fragment thereof. Preferably, the complex is aDOCK-AND-LOCK® (DNL®) complex, in which the components are joined usingthe binding affinity between a DDD (dimerization and docking domain)moiety of human protein kinase A (PKA) regulatory subunit RIα, RIβ, RIIαor RIIβ, and an AD (anchoring domain) moiety of an A-kinase anchoringprotein (AKAP), wherein a pair of DDD moieties forms a dimer that bindsto a complementary sequence on the AD moiety. Although the basic DNL®complex is trimeric, complexes with other stoichiometries are possible,such as tetrameric, pentameric or hexameric. The subject complexes areof use to treat autoimmune disease, inflammatory disease or otherconditions in which TNF-α and IL-6 play a pathogenic role. Inparticularly preferred embodiments, the disease or condition is selectedfrom the group consisting of systemic lupus erythematosus (SLE),rheumatoid arthritis, inflammatory bowel disease, type II diabetes,obesity, atherosclerosis and cachexia related to cancer.

BACKGROUND OF THE INVENTION

TNF-α and IL-6 are proinflammatory cytokines involved in thepathogenesis of various autoimmune diseases, such as rheumatoidarthritis (RA), systemic lupus erythematosus (SLE), inflammatory boweldisease, and type 2 diabetes. Blocking the biological activities ofTNF-α has demonstrated clinical benefits in patients with RA and Crohn'sdisease, as exemplified by five antibody- or receptor-based therapeuticscurrently on the market. The promise of IL-6 blockade was alsoreinforced by the regulatory approval of one anti-IL-6R antibody fortreating RA and juvenile idiopathic arthritis, with additionalantibodies targeting either IL-6R or IL-6 in advanced clinical trials.As reported by Mori et al. (Int Immunol 2011; 23: 701-12), IL-6 directlyactivates STAT3, whereas TNF-α indirectly activates STAT3 viastimulating the expression of IL-6, which then activates STAT3 andtriggers a cytokine amplification loop of IL-6, resulting in sustainedSTAT3 activation and chronic inflammation.

Numerous antibodies against TNF-α are commercially available and/orpublicly known, including infliximab (Jansenn Biotech, Inc.), adalimumab(Abbvie, Inc.), certolizumab pegol (UCB, Inc.) and golimumab (Centocor).Although these therapeutic agents have significantly improved thetreatment of certain autoimmune diseases, such as rheumatoid arthritis(RA), it has been reported that about 30% of RA patients treated withTNF inhibitors (including anti-TNFα antibodies) show little to no effectof the therapy, with about two thirds demonstrating moderate to highdisease activity at 1 year after treatment (Hirabara et al., 2014, ClinRheumatol 33:1247-54). Further, loss of therapeutic efficacy isfrequently observed with anti-TNF monoclonal antibodies (adalimumab,infliximab) in patients receiving concomitant low-dose methotrexate, dueto immunogenicity-related issues (Hirabara et al, 2014). A need existsfor more effective compositions and methods for use of anti-TNFantibodies in treating diseases and conditions related to TNF-α.

Dysregulated IL-6 production has been demonstrated to play apathological role in various autoimmune and chronic inflammatorydiseases. Therapies against IL-6 pathways have commonly targeted theIL-6 receptor (IL-6R), including the anti-IL-6R antibodies tocilizumab,and sarilumab. Antibodies targeted directly against IL-6 have also beendeveloped, such as olokizumab (UCB), siltuximab (Janssen), BMS-943429(Bristol-Myers Squibb) and sirukumab (Centocor). The latter have beenused against various autoimmune diseases and cancers. Followingregulatory approval of tocilizumab for rheumatoid arthritis, Castleman'sdisease and systemic juvenile idiopathic arthritis, favorable results ofoff-label use have been reported in systemic lupus erythematosus,systemic sclerosis, polymyositis, vasculitis syndrome including giantcell arteritis, Takayasu arteritis, cryoglobulinemia, glomerulonephritisand rheumatoid vasculitis (see, e.g., Tanaka & Kishimoto, 2012, Int JBiol Sci 8:1227-36). While these results are promising, no antibodiesagainst IL-6 (as opposed to IL-6R) have yet been approved for human usein any indication.

A need exists in the field for more effective, well-toleratedtherapeutic agents targeted against TNF and IL-6.

SUMMARY OF THE INVENTION

The present invention concerns compositions and methods of use ofbispecific or multispecific antibodies comprising at least oneanti-TNF-α antibody or antigen-binding fragment thereof and at least oneanti-IL-6 antibody or antigen-binding fragment thereof. Preferably, thebispecific or multispecific antibody is in the form of a DNL® complex,comprising AD and DDD moiety binding pairs as described below.

The antibodies may be chimeric, humanized or human antibodies. Incertain preferred embodiments, the antibodies are humanized, comprisingthe CDR sequences of, e.g., a murine anti-IL-6 or anti-TNF-α antibodyand the framework (FR) and constant region sequences from one or morehuman antibodies. Methods of antibody humanization are well known in theart, as discussed in detail below. The antibody can be of variousisotypes, preferably human IgG1, IgG2, IgG3 or IgG4, more preferablycomprising human IgG1 hinge and constant region sequences. Morepreferably, the antibody or fragment thereof may be designed or selectedto comprise human constant region sequences that belong to specificallotypes, which may result in reduced immunogenicity. Preferredallotypes for administration include a non-G1m1 allotype (nG1m1), suchas G1m3, G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype isselected from the group consisting of the nG1m1, G1m3, nG1m1,2 and Km3allotypes.

Numerous anti-TNF-α antibodies are commercially available and/orpublicly known, including but not limited to CDP571 (Ofei et al., 2011,Diabetes 45:881-85); MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B and M303(Thermo Scientific); 3H15L1, D13H3, TN3, 17H1L4, MP9-20A4, and 68B6A3 L1(Life Technologies); NBP1-19532, NB600-587, NBP2-27223, and NBP2-27224,(NOVUS BIOLOGICALS®); ab9635, (ABCAM®); certolizumab pegol (UCB,Brussels, Belgium); adalimumab (Abbvie); infliximab and golimumab(Centocor). These and many other known anti-TNF-α antibodies may be usedin the claimed methods and compositions.

Numerous anti-IL-6 antibodies are commercially available and/or publiclyknown, including but not limited to 5IL6, 4HCLC, 4H16L21, 677B6A2, and20F3 (Thermo Scientific); NBP1-47810, NBP2025275, NBP1047355, andNBP2021624 (NOVUS BIOLOGICALS®); olokizumab (UCB); siltuximab (Janssen);BMS-943429 (Bristol-Myers Squibb); and sirukumab (Centocor). These andmany other known anti-IL-6 antibodies may be used in the claimed methodsand compositions.

The subject antibodies may be co-administered with one or more othertherapeutic agents. The therapeutic agents may be conjugated to theantibodies or administered separately, either before, concomitantly withor after the antibody. Therapeutic agents of use for treating immune orinflammatory diseases are preferably selected from drugs,anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones,hormone antagonists, chemokines, prodrugs, enzymes, immunomodulators,cytokines or other known agents of use for immune or inflammatorydiseases.

Drugs of use may possess a pharmaceutical property selected from thegroup consisting of antimitotic, antikinase (e.g., anti-tyrosinekinase), alkylating, antimetabolite, antibiotic, alkaloid,anti-angiogenic, pro-apoptotic agents, immune modulators, andcombinations thereof.

Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine,anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine,docetaxel, dactinomycin, daunorubicin, doxorubicin,2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin,doxorubicin glucuronide, epirubicin glucuronide, estramustine,epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16),etoposide glucuronide, etoposide phosphate, floxuridine (FUdR),3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea,idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin,lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341,raloxifene, semustine, streptozocin, tamoxifen, temazolomide (an aqueousform of DTIC), transplatinum, thalidomide, thioguanine, thiotepa,teniposide, topotecan, uracil mustard, vinorelbine, vinblastine,vincristine and vinca alkaloids.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PlGFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -γ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT. Lenolidamide is yet another immunomodulator that has shownactivity in controlling certain cancers, such as multiple myeloma andhematopoietic tumors.

The antibodies or complexes may be used to treat a variety of diseasesor conditions in which TNF-α and IL-6 play a pathogenic role, such asautoimmune, immune dysfunction or inflammatory diseases. Exemplarydiseases or conditions may be selected from the group consisting ofrheumatoid arthritis (RA), systemic lupus erythematosus, type 2diabetes, Crohn's disease, Castleman's disease, juvenile idiopathicarthritis, systemic sclerosis, polymyositis, vasculitis syndrome,Takayasu arteritis, cryoglobulinemia, glomerulonephritis, rheumatoidvasculitis, arthritis, sepsis, septic shock, inflammation, non-septichyperinflammatory disorder, nephritis, inflammatory bowel disease,inflammatory liver injury, acute pancreatitis, acute respiratorydistress syndrome, ischemia-reperfusion injury, ischemic stroke,graft-vs.-host disease and cachexia related to cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Assay for neutralizing anti-IL-6 antibodies. Supernatants fromclones were incubated with human IL-6 at 37° C. for 1 hour, prior toincubation with HT-29 cells. The cells were incubated with rhIL-6 aloneor in combination with serum for 15 min at 37° C. and phosphorylation ofSTAT3 was detected by Western blotting.

FIG. 2A. Titration of neutralizing anti-IL-6 antibodies. The ability toblock IL-6 induced phosphorylation of STAT3 was determined by Westernblot analysis using the indicated concentrations of the 2-3B2 anti-IL-6antibody. A substantial inhibition of IL-6 dependent phosphorylation wasseen as low as 0.067 nM antibody.

FIG. 2B. Titration of neutralizing anti-IL-6 antibodies. The ability toblock IL-6 induced phosphorylation of STAT3 was determined by Westernblot analysis using the indicated concentrations of the 4-4E6 anti-IL-6antibody. Approximately equivalent effects on phosphorylation wereobserved at 0.67 nM 4-4E6 vs. 0.0067 nM 2-34B2 antibody (FIG. 2A).

FIG. 3. Neutralization activity of TNF-α mediated cytotoxicity byimmunized mouse sera on WEHI 164 cells. Serum from mouse #3 was the mosteffective at inhibiting TNF-α mediated cytotoxicity.

FIG. 4. Neutralization activity of TNF-α mediated cytotoxicity byantibodies from clones 4C9D11 and 4D3B11 in WEHI 164 cells.

FIG. 5. Neutralization activity of TNF-α mediated cytotoxicity byantibodies from clones 4C9D11G11 and 4D3B11C4 in L929 cells.

FIG. 6. Antibody-based neutralization of rhTNF-α-induced cell surfaceexpression of ICAM-1 in ECV-304 cells (a derivative of T24 bladdercancer cell line).

FIG. 7. Amino acid sequence of the anti-IL-6 antibody (2-3B2) heavychain (VH) sequence (SEQ ID NO:94). The sequence of a homologous heavychain of the B34781 antibody (SEQ ID NO:95), obtained from the NCBIprotein sequence database, is shown for comparison. Putative CDRsequences (underlined) were identified by comparison with the knownsequence of the homologous B34781 antibody.

FIG. 8. Amino acid sequence of the anti-IL-6 antibody (2-3B2) lightchain (VK) sequence (SEQ ID NO:96). The sequence of a homologous lightchain of AAB53778.1 (SEQ ID NO:97), obtained from the NCBI proteinsequence database, is shown for comparison. Putative CDR sequences(underlined) were identified by comparison with the known sequence ofthe homologous AAB53778.1.

FIG. 9. Activity of cIL6/TNFα DVD construct for neutralizing IL-6induced phosphorylation of STAT3 in HT-29 cells, compared to parent2-3B2 anti-IL-6 antibody.

FIG. 10. Amino acid sequence of the anti-TNF-α antibody (4C9) heavychain (VH) sequence (SEQ ID NO:98). The sequence of a homologous heavychain of the AAS66033.1 antibody (SEQ ID NO:99), obtained from the NCBIprotein sequence database, is shown for comparison. Putative CDRsequences (underlined) were identified by comparison with the knownsequence of the homologous AAS66033.1 antibody.

FIG. 11. Amino acid sequence of the anti-IL-6 antibody (4C9) light chain(VK) sequence (SEQ ID NO:100). The sequence of a homologous heavy chainof AAS66032.1 (SEQ ID NO:101), obtained from the NCBI protein sequencedatabase, is shown for comparison. Putative CDR sequences (underlined)were identified by comparison with the known sequence of the homologousAAS66032.1.

FIG. 12. Schematic illustration of the synthesis ofC_(K)-AD2-cIgG-anti-TNF-α-pdHL2.

FIG. 13. Inhibition of IL-6 induced phosphorylation of STAT3 bycT*-(c6)-(c6) complex compared to Fab-DDD2-cIL-6 protein.

FIG. 14. Inhibition of natural IL-6 induced phosphorylation of STAT3 bycT*-(c6)-(c6) complex compared to Fab-DDD2-cIL-6 protein.

FIG. 15. Inhibition of rhTNF-α induced cell death in L929 cells byanti-TNF-α antibody constructs.

FIG. 16. Inhibition of cell death induced by natural TNF-α in L929 cellsby anti-TNF-α antibody constructs.

FIG. 17. Relative affinities of cT*-(c6)-(c6), c-anti-TNF-α andc-anti-IL-6 for IL-6 and TNF-α from different species.

FIG. 18A. Role of STAT3 in IL-6 and TNF-α mediated pathways.

FIG. 18B. Role of STAT3 in IL-6 and TNF-α mediated disease processes.

DEFINITIONS

Unless otherwise specified, “a” or “an” means “one or more”.

As used herein, the terms “and” and “or” may be used to mean either theconjunctive or disjunctive. That is, both terms should be understood asequivalent to “and/or” unless otherwise stated.

A “therapeutic agent” is an atom, molecule, or compound that is usefulin the treatment of a disease. Examples of therapeutic agents includeantibodies, antibody fragments, peptides, drugs, toxins, enzymes,nucleases, hormones, immunomodulators, antisense oligonucleotides, smallinterfering RNA (siRNA), chelators, boron compounds, photoactive agents,dyes, and radioisotopes.

A “diagnostic agent” is an atom, molecule, or compound that is useful indiagnosing a disease. Useful diagnostic agents include, but are notlimited to, radioisotopes, dyes (such as with the biotin-streptavidincomplex), contrast agents, fluorescent compounds or molecules, andenhancing agents (e.g., paramagnetic ions) for magnetic resonanceimaging (MM).

An “antibody” as used herein refers to a full-length (i.e., naturallyoccurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment. An“antibody” includes monoclonal, polyclonal, bispecific, multispecific,murine, chimeric, humanized and human antibodies.

A “naked antibody” is an antibody or antigen binding fragment thereofthat is not attached to a therapeutic or diagnostic agent. The Fcportion of an intact naked antibody can provide effector functions, suchas complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev50:59-87, 1998). Other mechanisms by which naked antibodies induce celldeath may include apoptosis. (Vaswani and Hamilton, Ann Allergy AsthmaImmunol 81: 105-119, 1998.)

An “antibody fragment” is a portion of an intact antibody such asF(ab′)₂, F(ab)₂, Fab′, Fab, Fv, sFv, scFv, dAb and the like. Regardlessof structure, an antibody fragment binds with the same antigen that isrecognized by the full-length antibody. For example, antibody fragmentsinclude isolated fragments consisting of the variable regions, such asthe “Fv” fragments consisting of the variable regions of the heavy andlight chains or recombinant single chain polypeptide molecules in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”). “Single-chain antibodies”, often abbreviated as“scFv” consist of a polypeptide chain that comprises both a V_(H) and aV_(L) domain which interact to form an antigen-binding site. The V_(H)and V_(L) domains are usually linked by a peptide of 1 to 25 amino acidresidues. Antibody fragments also include diabodies, triabodies andsingle domain antibodies (dAb).

An antibody or antibody complex preparation, or a composition describedherein, is said to be administered in a “therapeutically effectiveamount” if the amount administered is physiologically significant. Anagent is physiologically significant if its presence results in adetectable change in the physiology of a recipient subject. Inparticular embodiments, an antibody preparation is physiologicallysignificant if its presence invokes an antitumor response or mitigatesthe signs and symptoms of an autoimmune disease state. A physiologicallysignificant effect could also be the evocation of a humoral and/orcellular immune response in the recipient subject leading to growthinhibition or death of target cells.

DOCK-AND-LOCK® (DNL®)

In preferred embodiments, a bivalent or multivalent antibody is formedas a DOCK-AND-LOCK® (DNL®) complex (see, e.g., U.S. Pat. Nos. 7,521,056;7,527,787; 7,534,866; 7,550,143 and 7,666,400, the Examples section ofeach of which is incorporated herein by reference.) Generally, thetechnique takes advantage of the specific and high-affinity bindinginteractions that occur between a dimerization and docking domain (DDD)sequence of the regulatory (R) subunits of cAMP-dependent protein kinase(PKA) and an anchor domain (AD) sequence derived from any of a varietyof AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wongand Scott, Nat. Rev. Mol. Cell Biol. 2004; 5: 959). The DDD and ADpeptides may be attached to any protein, peptide or other molecule.Because the DDD sequences spontaneously dimerize and bind to the ADsequence, the technique allows the formation of complexes between anyselected molecules that may be attached to DDD or AD sequences.

Although the standard DNL® complex comprises a trimer with twoDDD-linked molecules attached to one AD-linked molecule, variations incomplex structure allow the formation of dimers, trimers, tetramers,pentamers, hexamers and other multimers. In some embodiments, the DNL®complex may comprise two or more antibodies, antibody fragments orfusion proteins which bind to the same antigenic determinant or to twoor more different antigens. The DNL® complex may also comprise one ormore other effectors, such as proteins, peptides, immunomodulators,cytokines, interleukins, interferons, binding proteins, peptide ligands,carrier proteins, toxins, ribonucleases such as onconase, inhibitoryoligonucleotides such as siRNA, antigens or xenoantigens, polymers suchas PEG, enzymes, therapeutic agents, hormones, cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents or any other molecule oraggregate.

PKA, which plays a central role in one of the best studied signaltransduction pathways triggered by the binding of the second messengercAMP to the R subunits, was first isolated from rabbit skeletal musclein 1968 (Walsh et al., J. Biol. Chem. 1968; 243:3763). The structure ofthe holoenzyme consists of two catalytic subunits held in an inactiveform by the R subunits (Taylor, J. Biol. Chem. 1989; 264:8443). Isozymesof PKA are found with two types of R subunits (RI and RII), and eachtype has α and β isoforms (Scott, Pharmacol. Ther. 1991; 50:123). Thus,the four isoforms of PKA regulatory subunits are RIα, RIβ, RIIα andRIIβ. The R subunits have been isolated only as stable dimers and thedimerization domain has been shown to consist of the first 44amino-terminal residues of RIIα (Newlon et al., Nat. Struct. Biol. 1999;6:222). As discussed below, similar portions of the amino acid sequencesof other regulatory subunits are involved in dimerization and docking,each located near the N-terminal end of the regulatory subunit. Bindingof cAMP to the R subunits leads to the release of active catalyticsubunits for a broad spectrum of serine/threonine kinase activities,which are oriented toward selected substrates through thecompartmentalization of PKA via its docking with AKAPs (Scott et al., J.Biol. Chem. 1990; 265; 21561)

Since the first AKAP, microtubule-associated protein-2, wascharacterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984;81:6723), more than 50 AKAPs that localize to various sub-cellularsites, including plasma membrane, actin cytoskeleton, nucleus,mitochondria, and endoplasmic reticulum, have been identified withdiverse structures in species ranging from yeast to humans (Wong andScott, Nat. Rev. Mol. Cell Biol. 2004; 5:959). The AD of AKAPs for PKAis an amphipathic helix of 14-18 residues (Carr et al., J. Biol. Chem.1991; 266:14188). The amino acid sequences of the AD are quite variedamong individual AKAPs, with the binding affinities reported for RIIdimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA.2003; 100:4445). AKAPs will only bind to dimeric R subunits. For humanRIIα, the AD binds to a hydrophobic surface formed by the 23amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;6:216). Thus, the dimerization domain and AKAP binding domain of humanRIIα are both located within the same N-terminal 44 amino acid sequence(Newlon et al., Nat. Struct. Biol. 1999; 6:222; Newlon et al., EMBO J.2001; 20:1651), which is termed the DDD herein.

We have developed a platform technology to utilize the DDD of human PKAregulatory subunit RIα, RIβ, RIIα or RIIβ and the AD of AKAP as anexcellent pair of linker modules for docking any two entities, referredto hereafter as A and B, into a noncovalent complex, which could befurther locked into a DNL® complex through the introduction of cysteineresidues into both the DDD and AD at strategic positions to facilitatethe formation of disulfide bonds. The general methodology of theapproach is as follows. Entity A is constructed by linking a DDDsequence to a precursor of A, resulting in a first component hereafterreferred to as a. Because the DDD sequence would effect the spontaneousformation of a dimer, A would thus be composed of a₂. Entity B isconstructed by linking an AD sequence to a precursor of B, resulting ina second component hereafter referred to as b. The dimeric motif of DDDcontained in a₂ will create a docking site for binding to the ADsequence contained in b, thus facilitating a ready association of a₂ andb to form a binary, trimeric complex composed of a₂b. This binding eventis made irreversible with a subsequent reaction to covalently secure thetwo entities via disulfide bridges, which occurs very efficiently basedon the principle of effective local concentration because the initialbinding interactions should bring the reactive thiol groups placed ontoboth the DDD and AD into proximity (Chmura et al., Proc. Natl. Acad.Sci. USA. 2001; 98:8480) to ligate site-specifically. Using variouscombinations of linkers, adaptor modules and precursors, a wide varietyof DNL® constructs of different stoichiometry may be produced and used(see, e.g., U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866; 7,527,787and 7,666,400.)

By attaching the DDD and AD away from the functional groups of the twoprecursors, such site-specific ligations are also expected to preservethe original activities of the two precursors. This approach is modularin nature and potentially can be applied to link, site-specifically andcovalently, a wide range of substances, including peptides, proteins,antibodies, antibody fragments, and other effector moieties with a widerange of activities. Utilizing the fusion protein method of constructingAD and DDD conjugated effectors described in the Examples below,virtually any protein or peptide may be incorporated into a DNL®construct. However, the technique is not limiting and other methods ofconjugation may be utilized.

A variety of methods are known for making fusion proteins, includingnucleic acid synthesis, hybridization and/or amplification to produce asynthetic double-stranded nucleic acid encoding a fusion protein ofinterest. Such double-stranded nucleic acids may be inserted intoexpression vectors for fusion protein production by standard molecularbiology techniques (see, e.g. Sambrook et al., Molecular Cloning, Alaboratory manual, 2^(nd) Ed, 1989). In such preferred embodiments, theAD and/or DDD moiety may be attached to either the N-terminal orC-terminal end of an effector protein or peptide. However, the skilledartisan will realize that the site of attachment of an AD or DDD moietyto an effector moiety may vary, depending on the chemical nature of theeffector moiety and the part(s) of the effector moiety involved in itsphysiological activity. Site-specific attachment of a variety ofeffector moieties may be performed using techniques known in the art,such as the use of bivalent cross-linking reagents and/or other chemicalconjugation techniques.

Structure-Function Relationships in AD and DDD Moieties

For different types of DNL® constructs, different AD or DDD sequencesmay be utilized. Exemplary DDD and AD sequences are provided below.

DDD1 (SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA DDD2(SEQ ID NO: 2) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA AD1(SEQ ID NO: 3) QIEYLAKQIVDNAIQQA AD2 (SEQ ID NO: 4)CGQIEYLAKQIVDNAIQQAGC

The skilled artisan will realize that DDD1 and DDD2 are based on the DDDsequence of the human RIIα isoform of protein kinase A. However, inalternative embodiments, the DDD and AD moieties may be based on the DDDsequence of the human RIα form of protein kinase A and a correspondingAKAP sequence, as exemplified in DDD3, DDD3C and AD3 below.

DDD3 (SEQ ID NO: 5) SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAKDDD3C (SEQ ID NO: 6) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK AD3 (SEQ ID NO: 7) CGFEELAWKIAKMIWSDVFQQGC

In other alternative embodiments, other sequence variants of AD and/orDDD moieties may be utilized in construction of the DNL® complexes. Forexample, there are only four variants of human PKA DDD sequences,corresponding to the DDD moieties of PKA RIα, RIIα, RIβ and RIIβ. TheRIIα DDD sequence is the basis of DDD1 and DDD2 disclosed above. Thefour human PKA DDD sequences are shown below. The DDD sequencerepresents residues 1-44 of RIIα, 1-44 of RIIβ, 12-61 of RIα and 13-66of RIβ. (Note that the sequence of DDD1 is modified slightly from thehuman PKA RIIα DDD moiety.)

PKA RIα (SEQ ID NO: 8) SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK PKA RIβ (SEQ ID NO: 9)SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEEN RQILA PKA RIIα(SEQ ID NO: 10) SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ PKA RIIβ(SEQ ID NO: 11) SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER

The structure-function relationships of the AD and DDD domains have beenthe subject of investigation. (See, e.g., Burns-Hamuro et al., 2005,Protein Sci 14:2982-92; Carr et al., 2001, J Biol Chem 276:17332-38;Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker etal., 2006, Biochem J 396:297-306; Stokka et al., 2006, Biochem J400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al.,2006, Mol Cell 24:397-408, the entire text of each of which isincorporated herein by reference.)

For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined thecrystal structure of the AD-DDD binding interaction and concluded thatthe human DDD sequence contained a number of conserved amino acidresidues that were important in either dimer formation or AKAP binding,underlined in SEQ ID NO:1 below. (See FIG. 1 of Kinderman et al., 2006,incorporated herein by reference.) The skilled artisan will realize thatin designing sequence variants of the DDD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical fordimerization and AKAP binding.

(SEQ ID NO: 1) SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

As discussed in more detail below, conservative amino acid substitutionshave been characterized for each of the twenty common L-amino acids.Thus, based on the data of Kinderman (2006) and conservative amino acidsubstitutions, potential alternative DDD sequences based on SEQ ID NO:1are shown in Table 1. In devising Table 1, only highly conservativeamino acid substitutions were considered. For example, charged residueswere only substituted for residues of the same charge, residues withsmall side chains were substituted with residues of similar size,hydroxyl side chains were only substituted with other hydroxyls, etc.Because of the unique effect of proline on amino acid secondarystructure, no other residues were substituted for proline. A limitednumber of such potential alternative DDD moiety sequences are shown inSEQ ID NO:12 to SEQ ID NO:31 below. The skilled artisan will realizethat an almost unlimited number of alternative species within the genusof DDD moieties can be constructed by standard techniques, for exampleusing a commercial peptide synthesizer or well known site-directedmutagenesis techniques. The effect of the amino acid substitutions on ADmoiety binding may also be readily determined by standard bindingassays, for example as disclosed in Alto et al. (2003, Proc Natl AcadSci USA 100:4445-50).

TABLE 1 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO: 1).Consensus sequence disclosed as SEQ ID NO: 87. S H I Q I P P G L T E L LQ G Y T V E V L R T K N A S D N A S D K R Q Q P P D L V E F A V E Y F TR L R E A R A N N E D L D S K K D L K L I I I V V VTHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 12)SKIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 13)SRIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 14)SHINIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 15)SHIQIPPALTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 16)SHIQIPPGLSELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 17)SHIQIPPGLTDLLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO :18)SHIQIPPGLTELLNGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 19)SHIQIPPGLTELLQAYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 20)SHIQIPPGLTELLQGYSVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 21)SHIQIPPGLTELLQGYTVDVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 22)SHIQIPPGLTELLQGYTVEVLKQQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 23)SHIQIPPGLTELLQGYTVEVLRNQPPDLVEFAVEYFTRLREARA (SEQ ID NO: 24)SHIQIPPGLTELLQGYTVEVLRQNPPDLVEFAVEYFTRLREARA (SEQ ID NO: 25)SHIQIPPGLTELLQGYTVEVLRQQPPELVEFAVEYFTRLREARA (SEQ ID NO: 26)SHIQIPPGLTELLQGYTVEVLRQQPPDLVDFAVEYFTRLREARA (SEQ ID NO: 27)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFLVEYFTRLREARA (SEQ ID NO: 28)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFIVEYFTRLREARA (SEQ ID NO: 29)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFVVEYFTRLREARA (SEQ ID NO: 30)SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVDYFTRLREARA (SEQ ID NO: 31)

Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed abioinformatic analysis of the AD sequence of various AKAP proteins todesign an RII selective AD sequence called AKAP-IS (SEQ ID NO:3), with abinding constant for DDD of 0.4 nM. The AKAP-IS sequence was designed asa peptide antagonist of AKAP binding to PKA. Residues in the AKAP-ISsequence where substitutions tended to decrease binding to DDD areunderlined in SEQ ID NO:3 below. The skilled artisan will realize thatin designing sequence variants of the AD sequence, one would desirablyavoid changing any of the underlined residues, while conservative aminoacid substitutions might be made for residues that are less critical forDDD binding. Table 2 shows potential conservative amino acidsubstitutions in the sequence of AKAP-IS (AD1, SEQ ID NO:3), similar tothat shown for DDD1 (SEQ ID NO:1) in Table 1 above.

A limited number of such potential alternative AD moiety sequences areshown in SEQ ID NO:32 to SEQ ID NO:49 below. Again, a very large numberof species within the genus of possible AD moiety sequences could bemade, tested and used by the skilled artisan, based on the data of Altoet al. (2003). It is noted that FIG. 2 of Alto (2003) shows an evenlarge number of potential amino acid substitutions that may be made,while retaining binding activity to DDD moieties, based on actualbinding experiments.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

TABLE 2 Conservative Amino Acid Substitutions in AD1 (SEQ ID NO: 3).Consensus sequence disclosed as SEQ ID NO: 88. Q I E Y L A K Q I V D N AI Q Q A N L D F I R N E Q N N L V T V I S VNIEYLAKQIVDNAIQQA (SEQ ID NO: 32) QLEYLAKQIVDNAIQQA (SEQ ID NO: 33)QVEYLAKQIVDNAIQQA (SEQ ID NO: 34) QIDYLAKQIVDNAIQQA (SEQ ID NO: 35)QIEFLAKQIVDNAIQQA (SEQ ID NO: 36) QIETLAKQIVDNAIQQA (SEQ ID NO: 37)QIESLAKQIVDNAIQQA (SEQ ID NO: 38) QIEYIAKQIVDNAIQQA (SEQ ID NO: 39)QIEYVAKQIVDNAIQQA (SEQ ID NO: 40) QIEYLARQIVDNAIQQA (SEQ ID NO: 41)QIEYLAKNIVDNAIQQA (SEQ ID NO: 42) QIEYLAKQIVENAIQQA (SEQ ID NO: 43)QIEYLAKQIVDQAIQQA (SEQ ID NO: 44) QIEYLAKQIVDNAINQA (SEQ ID NO: 45)QIEYLAKQIVDNAIQNA (SEQ ID NO: 46) QIEYLAKQIVDNAIQQL (SEQ ID NO: 47)QIEYLAKQIVDNAIQQI (SEQ ID NO: 48) QIEYLAKQIVDNAIQQV (SEQ ID NO: 49)

Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography andpeptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:50),exhibiting a five order of magnitude higher selectivity for the RIIisoform of PKA compared with the RI isoform. Underlined residuesindicate the positions of amino acid substitutions, relative to theAKAP-IS sequence, which increased binding to the DDD moiety of RIIα. Inthis sequence, the N-terminal Q residue is numbered as residue number 4and the C-terminal A residue is residue number 20. Residues wheresubstitutions could be made to affect the affinity for RIIα wereresidues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It iscontemplated that in certain alternative embodiments, the SuperAKAP-ISsequence may be substituted for the AKAP-IS AD moiety sequence toprepare DNL® constructs. Other alternative sequences that might besubstituted for the AKAP-IS AD sequence are shown in SEQ ID NO:51-53.Substitutions relative to the AKAP-IS sequence are underlined. It isanticipated that, as with the AD2 sequence shown in SEQ ID NO:4, the ADmoiety may also include the additional N-terminal residues cysteine andglycine and C-terminal residues glycine and cysteine.

SuperAKAP-IS (SEQ ID NO: 50) QIEYVAKQIVDYAIHQAAlternative AKAP sequences (SEQ ID NO: 51) QIEYKAKQIVDHAIHQA(SEQ ID NO: 52) QIEYHAKQIVDHAIHQA (SEQ ID NO: 53) QIEYVAKQIVDHAIHQA

FIG. 2 of Gold et al. disclosed additional DDD-binding sequences from avariety of AKAP proteins, shown below.

Rh-Specific AKAPs AKAP-KL (SEQ ID NO: 54) PLEYQAGLLVQNAIQQAI AKAP 79(SEQ ID NO: 55) LLIETASSLVKNAIQLSI AKAP-Lbc (SEQ ID NO: 56)LIEEAASRIVDAVIEQVK RI-Specific AKAPs AKAPce (SEQ ID NO: 57)ALYQFADRFSELVISEAL RIAD (SEQ ID NO: 58) LEQVANQLADQIIKEAT PV38(SEQ ID NO: 59) FEELAWKIAKMIWSDVF Dual-Specificity AKAPs AKAP 7(SEQ ID NO: 60) ELVRLSKRLVENAVLKAV MAP2D (SEQ ID NO: 61)TAEEVSARIVQVVTAEAV DAKAP 1 (SEQ ID NO: 62) QIKQAAFQLISQVILEAT DAKAP 2(SEQ ID NO: 63) LAWKIAKMIVSDVMQQ

Stokka et al. (2006, Biochem J 400:493-99) also developed peptidecompetitors of AKAP binding to PKA, shown in SEQ ID NO:64-66. Thepeptide antagonists were designated as Ht31 (SEQ ID NO:64), RIAD (SEQ IDNO:65) and PV-38 (SEQ ID NO:66). The Ht-31 peptide exhibited a greateraffinity for the RII isoform of PKA, while the RIAD and PV-38 showedhigher affinity for RI.

Ht31 (SEQ ID NO: 64) DLIEEAASRIVDAVIEQVKAAGAY RIAD (SEQ ID NO: 65)LEQYANQLADQIIKEATE PV-38 (SEQ ID NO: 66) FEELAWKIAKMIWSDVFQQC

Hundsrucker et al. (2006, Biochem J 396:297-306) developed still otherpeptide competitors for AKAP binding to PKA, with a binding constant aslow as 0.4 nM to the DDD of the RII form of PKA. The sequences ofvarious AKAP antagonistic peptides are provided in Table 1 ofHundsrucker et al., reproduced in Table 3 below. AKAPIS represents asynthetic RII subunit-binding peptide. All other peptides are derivedfrom the RII-binding domains of the indicated AKAPs.

TABLE 3 AKAP Peptide sequences Peptide Sequence AKAPISQIEYLAKQIVDNAIQQA (SEQ ID NO: 3) AKAPIS-PQIEYLAKQIPDNAIQQA (SEQ ID NO: 67) Ht31KGADLIEEAASRIVDAVIEQVKAAG (SEQ ID NO: 68) Ht31-PKGADLIEEAASRIPDAPIEQVKAAG (SEQ ID NO: 69) AKAP7δ-wt-pepPEDAELVRLSKRLVENAVLKAVQQY (SEQ ID NO: 70) AKAP7δ-L304T-pepPEDAELVRTSKRLVENAVLKAVQQY (SEQ ID NO: 71) AKAP7δ-L308D-pepPEDAELVRLSKRDVENAVLKAVQQY (SEQ ID NO: 72) AKAP7δ-P-pepPEDAELVRLSKRLPENAVLKAVQQY (SEQ ID NO: 73) AKAP7δ-PP-pepPEDAELVRLSKRLPENAPLKAVQQY (SEQ ID NO: 74) AKAP7δ-L314E-pepPEDAELVRLSKRLVENAVEKAVQQY (SEQ ID NO: 75) AKAP1-pepEEGLDRNEEIKRAAFQIISQVISEA (SEQ ID NO: 76) AKAP2-pepLVDDPLEYQAGLLVQNAIQQAIAEQ (SEQ ID NO: 77) AKAP5-pepQYETLLIETASSLVKNAIQLSIEQL (SEQ ID NO: 78) AKAP9-pepLEKQYQEQLEEEVAKVIVSMSIAFA (SEQ ID NO: 79) AKAP10-pepNTDEAQEELAWKIAKMIVSDIMQQA (SEQ ID NO: 80) AKAP11-pepVNLDKKAVLAEKIVAEAIEKAEREL (SEQ ID NO: 81) AKAP12-pepNGILELETKSSKLVQNIIQTAVDQF (SEQ ID NO: 82) AKAP14-pepTQDKNYEDELTQVALALVEDVINYA (SEQ ID NO: 83) Rab32-pepETSAKDNINIEEAARFLVEKILVNH (SEQ ID NO: 84)

Residues that were highly conserved among the AD domains of differentAKAP proteins are indicated below by underlining with reference to theAKAP IS sequence (SEQ ID NO:3). The residues are the same as observed byAlto et al. (2003), with the addition of the C-terminal alanine residue.(See FIG. 4 of Hundsrucker et al. (2006), incorporated herein byreference.) The sequences of peptide antagonists with particularly highaffinities for the RII DDD sequence were those of AKAP-IS,AKAP7δ-wt-pep, AKAP7δ-L304T-pep and AKAP7δ-L308D-pep.

AKAP-IS (SEQ ID NO: 3) QIEYLAKQIVDNAIQQA

Carr et al. (2001, J Biol Chem 276:17332-38) examined the degree ofsequence homology between different AKAP-binding DDD sequences fromhuman and non-human proteins and identified residues in the DDDsequences that appeared to be the most highly conserved among differentDDD moieties. These are indicated below by underlining with reference tothe human PKA RIIα DDD sequence of SEQ ID NO:1. Residues that wereparticularly conserved are further indicated by italics. The residuesoverlap with, but are not identical to those suggested by Kinderman etal. (2006) to be important for binding to AKAP proteins. The skilledartisan will realize that in designing sequence variants of DDD, itwould be most preferred to avoid changing the most conserved residues(italicized), and it would be preferred to also avoid changing theconserved residues (underlined), while conservative amino acidsubstitutions may be considered for residues that are neither underlinednor italicized.

(SEQ ID NO: 1) SHIQ I PPG L TE LL QG Y T V E VLRQQPPD LVE F A V E YFTR LREARA

A modified set of conservative amino acid substitutions for the DDD1(SEQ ID NO:1) sequence, based on the data of Carr et al. (2001) is shownin Table 4. Even with this reduced set of substituted sequences, thereare over 65,000 possible alternative DDD moiety sequences that may beproduced, tested and used by the skilled artisan without undueexperimentation. The skilled artisan could readily derive suchalternative DDD amino acid sequences as disclosed above for Table 1 andTable 2.

TABLE 4 Conservative Amino Acid Substitutions in DDD1 (SEQ ID NO:1).Consensus sequence disclosed as SEQ ID NO: 89. S H I Q I P P G L T E L LQ G Y T V E V L R T N S I L A Q Q P P D L V E F A V E Y F T R L R E A RA N I D S K K L L L I I A  V V

The skilled artisan will realize that these and other amino acidsubstitutions in the DDD or AD amino acid sequences may be utilized toproduce alternative species within the genus of AD or DDD moieties,using techniques that are standard in the field and only routineexperimentation.

Amino Acid Substitutions

In alternative embodiments, the disclosed methods and compositions mayinvolve production and use of proteins or peptides with one or moresubstituted amino acid residues. For example, the DDD and/or ADsequences used to make DNL® constructs may be modified as discussedabove.

The skilled artisan will be aware that, in general, amino acidsubstitutions typically involve the replacement of an amino acid withanother amino acid of relatively similar properties (i.e., conservativeamino acid substitutions). The properties of the various amino acids andeffect of amino acid substitution on protein structure and function havebeen the subject of extensive study and knowledge in the art.

For example, the hydropathic index of amino acids may be considered(Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relativehydropathic character of the amino acid contributes to the secondarystructure of the resultant protein, which in turn defines theinteraction of the protein with other molecules. Each amino acid hasbeen assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte & Doolittle, 1982), these are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5). In making conservative substitutions, the use of amino acidswhose hydropathic indices are within ±2 is preferred, within ±1 are morepreferred, and within ±0.5 are even more preferred.

Amino acid substitution may also take into account the hydrophilicity ofthe amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5.+−.1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Replacement ofamino acids with others of similar hydrophilicity is preferred.

Other considerations include the size of the amino acid side chain. Forexample, it would generally not be preferred to replace an amino acidwith a compact side chain, such as glycine or serine, with an amino acidwith a bulky side chain, e.g., tryptophan or tyrosine. The effect ofvarious amino acid residues on protein secondary structure is also aconsideration. Through empirical study, the effect of different aminoacid residues on the tendency of protein domains to adopt analpha-helical, beta-sheet or reverse turn secondary structure has beendetermined and is known in the art (see, e.g., Chou & Fasman, 1974,Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979,Biophys. J., 26:367-384).

Based on such considerations and extensive empirical study, tables ofconservative amino acid substitutions have been constructed and areknown in the art. For example: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R)gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys(C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H)asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met,ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F)leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W)phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

Other considerations for amino acid substitutions include whether or notthe residue is located in the interior of a protein or is solventexposed. For interior residues, conservative substitutions wouldinclude: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala andGly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr;Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solventexposed residues, conservative substitutions would include: Asp and Asn;Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala andGly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu;Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have beenconstructed to assist in selection of amino acid substitutions, such asthe PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlanmatrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix,Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)

In determining amino acid substitutions, one may also consider theexistence of intermolecular or intramolecular bonds, such as formationof ionic bonds (salt bridges) between positively charged residues (e.g.,His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) ordisulfide bonds between nearby cysteine residues.

Methods of substituting any amino acid for any other amino acid in anencoded protein sequence are well known and a matter of routineexperimentation for the skilled artisan, for example by the technique ofsite-directed mutagenesis or by synthesis and assembly ofoligonucleotides encoding an amino acid substitution and splicing intoan expression vector construct.

Antibodies and Antibody Fragments

Techniques for preparing monoclonal antibodies against virtually anytarget antigen, such as IL-6 or TNF-α, are well known in the art. See,for example, Kohler and Milstein, Nature 256: 495 (1975), and Coligan etal. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7(John Wiley & Sons 1991). Briefly, monoclonal antibodies can be obtainedby injecting mice with a composition comprising an antigen, removing thespleen to obtain B-lymphocytes, fusing the B-lymphocytes with myelomacells to produce hybridomas, cloning the hybridomas, selecting positiveclones which produce antibodies to the antigen, culturing the clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

MAbs can be isolated and purified from hybridoma cultures by a varietyof well-established techniques. Such isolation techniques includeaffinity chromatography with Protein-A SEPHAROSE®, size-exclusionchromatography, and ion-exchange chromatography. See, for example,Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines etal., “Purification of Immunoglobulin G (IgG),” in METHODS IN MOLECULARBIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).

After the initial raising of antibodies to the immunogen, the antibodiescan be sequenced and subsequently prepared by recombinant techniques.Humanization and chimerization of murine antibodies and antibodyfragments are well known to those skilled in the art. The use ofantibody components derived from humanized, chimeric or human antibodiesobviates potential problems associated with the immunogenicity of murineconstant regions.

Chimeric Antibodies

A chimeric antibody is a recombinant protein in which the variableregions of a human antibody have been replaced by the variable regionsof, for example, a mouse antibody, including thecomplementarity-determining regions (CDRs) of the mouse antibody.Chimeric antibodies exhibit decreased immunogenicity and increasedstability when administered to a subject. General techniques for cloningmurine immunoglobulin variable domains are disclosed, for example, inOrlandi et al., Proc. Nat'l Acad. Sci. USA 86: 3833 (1989). Techniquesfor constructing chimeric antibodies are well known to those of skill inthe art. As an example, Leung et al., Hybridoma 13:469 (1994), producedan LL2 chimera by combining DNA sequences encoding the V_(κ) and V_(H)domains of murine LL2, an anti-CD22 monoclonal antibody, with respectivehuman κ and IgG₁ constant region domains.

Humanized Antibodies

Techniques for producing humanized MAbs are well known in the art (see,e.g., Jones et al., Nature 321: 522 (1986), Riechmann et al., Nature332: 323 (1988), Verhoeyen et al., Science 239: 1534 (1988), Carter etal., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev.Biotech. 12: 437 (1992), and Singer et al., J. Immun. 150: 2844 (1993)).A chimeric or murine monoclonal antibody may be humanized bytransferring the mouse CDRs from the heavy and light variable chains ofthe mouse immunoglobulin into the corresponding variable domains of ahuman antibody. The mouse framework regions (FR) in the chimericmonoclonal antibody are also replaced with human FR sequences. As simplytransferring mouse CDRs into human FRs often results in a reduction oreven loss of antibody affinity, additional modification might berequired in order to restore the original affinity of the murineantibody. This can be accomplished by the replacement of one or morehuman residues in the FR regions with their murine counterparts toobtain an antibody that possesses good binding affinity to its epitope.See, for example, Tempest et al., Biotechnology 9:266 (1991) andVerhoeyen et al., Science 239: 1534 (1988). Generally, those human FRamino acid residues that differ from their murine counterparts and arelocated close to or touching one or more CDR amino acid residues wouldbe candidates for substitution.

Human Antibodies

Methods for producing fully human antibodies using either combinatorialapproaches or transgenic animals transformed with human immunoglobulinloci are known in the art (e.g., Mancini et al., 2004, New Microbiol.27:315-28; Conrad and Scheller, 2005, Comb. Chem. High ThroughputScreen. 8:117-26; Brekke and Loset, 2003, Curr. Opin. Phamacol.3:544-50). A fully human antibody also can be constructed by genetic orchromosomal transfection methods, as well as phage display technology,all of which are known in the art. See for example, McCafferty et al.,Nature 348:552-553 (1990). Such fully human antibodies are expected toexhibit even fewer side effects than chimeric or humanized antibodiesand to function in vivo as essentially endogenous human antibodies. Incertain embodiments, the claimed methods and procedures may utilizehuman antibodies produced by such techniques.

In one alternative, the phage display technique may be used to generatehuman antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mol. Res.4:126-40). Human antibodies may be generated from normal humans or fromhumans that exhibit a particular disease state, such as cancer(Dantas-Barbosa et al., 2005). The advantage to constructing humanantibodies from a diseased individual is that the circulating antibodyrepertoire may be biased towards antibodies against disease-associatedantigens.

In one non-limiting example of this methodology, Dantas-Barbosa et al.(2005) constructed a phage display library of human Fab antibodyfragments from osteosarcoma patients. Generally, total RNA was obtainedfrom circulating blood lymphocytes (Id.). Recombinant Fab were clonedfrom the μ, γ and κ chain antibody repertoires and inserted into a phagedisplay library (Id.). RNAs were converted to cDNAs and used to make FabcDNA libraries using specific primers against the heavy and light chainimmunoglobulin sequences (Marks et al., 1991, J. Mol. Biol. 222:581-97).Library construction was performed according to Andris-Widhopf et al.(2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 1^(st)edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.pp. 9.1 to 9.22). The final Fab fragments were digested with restrictionendonucleases and inserted into the bacteriophage genome to make thephage display library. Such libraries may be screened by standard phagedisplay methods, as known in the art (see, e.g., Pasqualini andRuoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J.Nucl. Med. 43:159-162).

Phage display can be performed in a variety of formats, for theirreview, see e.g. Johnson and Chiswell, Current Opinion in StructuralBiology 3:5564-571 (1993). Human antibodies may also be generated by invitro activated B cells. See U.S. Pat. Nos. 5,567,610 and 5,229,275,incorporated herein by reference in their entirety. The skilled artisanwill realize that these techniques are exemplary and any known methodfor making and screening human antibodies or antibody fragments may beutilized.

In another alternative, transgenic animals that have been geneticallyengineered to produce human antibodies may be used to generateantibodies against essentially any immunogenic target, using standardimmunization protocols. Methods for obtaining human antibodies fromtransgenic mice are disclosed by Green et al., Nature Genet. 7:13(1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int.Immun. 6:579 (1994). A non-limiting example of such a system is theXENOMOUSE® (e.g., Green et al., 1999, J. Immunol. Methods 231:11-23)from Abgenix (Fremont, Calif.). In the XENOMOUSE® and similar animals,the mouse antibody genes have been inactivated and replaced byfunctional human antibody genes, while the remainder of the mouse immunesystem remains intact.

The XENOMOUSE® was transformed with germline-configured YACs (yeastartificial chromosomes) that contained portions of the human IgH andIgkappa loci, including the majority of the variable region sequences,along accessory genes and regulatory sequences. The human variableregion repertoire may be used to generate antibody producing B cells,which may be processed into hybridomas by known techniques. A XENOMOUSE®immunized with a target antigen will produce human antibodies by thenormal immune response, which may be harvested and/or produced bystandard techniques discussed above. A variety of strains of XENOMOUSE®are available, each of which is capable of producing a different classof antibody. Transgenically produced human antibodies have been shown tohave therapeutic potential, while retaining the pharmacokineticproperties of normal human antibodies (Green et al., 1999). The skilledartisan will realize that the claimed compositions and methods are notlimited to use of the XENOMOUSE® system but may utilize any transgenicanimal that has been genetically engineered to produce human antibodies.

Antibody Fragments

Antibody fragments which recognize specific epitopes can be generated byknown techniques. Antibody fragments are antigen binding portions of anantibody, such as F(ab′)₂, Fab′, F(ab)₂, Fab, Fv, sFv and the like.F(ab′)₂ fragments can be produced by pepsin digestion of the antibodymolecule and Fab′ fragments can be generated by reducing disulfidebridges of the F(ab′)₂ fragments. Alternatively, Fab′ expressionlibraries can be constructed (Huse et al., 1989, Science, 246:1274-1281)to allow rapid and easy identification of monoclonal Fab′ fragments withthe desired specificity. F(ab)₂ fragments may be generated by papaindigestion of an antibody.

A single chain Fv molecule (scFv) comprises a VL domain and a VH domain.The VL and VH domains associate to form a target binding site. These twodomains are further covalently linked by a peptide linker (L). Methodsfor making scFv molecules and designing suitable peptide linkers aredescribed in U.S. Pat. No. 4,704,692, U.S. Pat. No. 4,946,778, R. Raagand M. Whitlow, “Single Chain Fvs.” FASEB Vol 9:73-80 (1995) and R. E.Bird and B. W. Walker, “Single Chain Antibody Variable Regions,”TIBTECH, Vol 9: 132-137 (1991).

Techniques for producing single domain antibodies are also known in theart, as disclosed for example in Cossins et al. (2006, Prot ExpressPurif 51:253-259), incorporated herein by reference. Single domainantibodies (VHH) may be obtained, for example, from camels, alpacas orllamas by standard immunization techniques. (See, e.g., Muyldermans etal., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75,2003; Maass et al., J Immunol Methods 324:13-25, 2007). The VHH may havepotent antigen-binding capacity and can interact with novel epitopesthat are inaccessible to conventional VH-VL pairs. (Muyldermans et al.,2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgGantibodies (HCAbs) (Maass et al., 2007). Alpacas may be immunized withknown antigens, such as TNF-α, and VHHs can be isolated that bind to andneutralize the target antigen (Maass et al., 2007). PCR primers thatamplify virtually all alpaca VHH coding sequences have been identifiedand may be used to construct alpaca VHH phage display libraries, whichcan be used for antibody fragment isolation by standard biopanningtechniques well known in the art (Maass et al., 2007). In certainembodiments, anti-pancreatic cancer VHH antibody fragments may beutilized in the claimed compositions and methods.

An antibody fragment can be prepared by proteolytic hydrolysis of thefull length antibody or by expression in E. coli or another host of theDNA coding for the fragment. An antibody fragment can be obtained bypepsin or papain digestion of full length antibodies by conventionalmethods. These methods are described, for example, by Goldenberg, U.S.Pat. Nos. 4,036,945 and 4,331,647 and references contained therein.Also, see Nisonoff et al., Arch Biochem. Biophys. 89: 230 (1960);Porter, Biochem. J. 73: 119 (1959), Edelman et al., in METHODS INENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages2.8.1-2.8.10 and 2.10.-2.10.4.

Known Antibodies

Various embodiments, for example in combination therapy, may involve theuse of antibodies binding to target antigens besides IL-6 or TNF-α. Avariety of antibodies are commercially available and/or known in theart. Antibodies of use may be commercially obtained, for example, fromthe American Type Culture Collection (ATCC, Manassas, Va.). A largenumber of antibodies against various disease targets, including but notlimited to tumor-associated antigens, have been deposited at the ATCCand/or have published variable region sequences and are available foruse in the claimed methods and compositions. See, e.g., U.S. Pat. Nos.7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;7,049,060; 7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018;7,037,498; 7,012,133; 7,001,598; 6,998,468; 6,994,976; 6,994,852;6,989,241; 6,974,863; 6,965,018; 6,964,854; 6,962,981; 6,962,813;6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;6,921,645; 6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475;6,905,681; 6,899,879; 6,893,625; 6,887,468; 6,887,466; 6,884,594;6,881,405; 6,878,812; 6,875,580; 6,872,568; 6,867,006; 6,864,062;6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;6,824,780; 6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450;6,767,711; 6,764,688; 6,764,681; 6,764,679; 6,743,898; 6,733,981;6,730,307; 6,720,155; 6,716,966; 6,709,653; 6,693,176; 6,692,908;6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;6,673,344; 6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833;6,610,294; 6,605,441; 6,605,279; 6,596,852; 6,592,868; 6,576,745;6,572,856; 6,566,076; 6,562,618; 6,545,130; 6,544,749; 6,534,058;6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;6,488,930; 6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529;6,465,173; 6,461,823; 6,458,356; 6,455,044; 6,455,040, 6,451,310;6,444,206; 6,441,143; 6,432,404; 6,432,402; 6,419,928; 6,413,726;6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;6,383,759; 6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481;6,355,444; 6,355,245; 6,355,244; 6,346,246; 6,344,198; 6,340,571;6,340,459; 6,331,175; 6,306,393; 6,254,868; 6,187,287; 6,183,744;6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;5,814,440; 5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595;5,677,136; 5,587,459; 5,443,953, 5,525,338, the Examples section of eachof which is incorporated herein by reference. These are exemplary onlyand a wide variety of other antibodies and their hybridomas are known inthe art. The skilled artisan will realize that antibody sequences orantibody-secreting hybridomas against almost any disease-associatedantigen may be obtained by a simple search of the ATCC, NCBI and/orUSPTO databases for antibodies against a selected disease-associatedtarget of interest. The antigen binding domains of the cloned antibodiesmay be amplified, excised, ligated into an expression vector,transfected into an adapted host cell and used for protein production,using standard techniques well known in the art (see, e.g., U.S. Pat.Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples sectionof each of which is incorporated herein by reference).

Particular antibodies that may be of use for therapy of cancer withinthe scope of the claimed methods and compositions include, but are notlimited to, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), RS7(anti-epithelial glycoprotein-1 (EGP-1)), PAM4 and KC4 (bothanti-mucin), MN-14 (anti-carcinoembryonic antigen (CEA, also known asCD66e), Mu-9 (anti-colon-specific antigen-p), Immu 31 (ananti-alpha-fetoprotein), TAG-72 (e.g., CC49), Tn, J591 or HuJ591(anti-PSMA (prostate-specific membrane antigen)), AB-PG1-XG1-026(anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX),hL243 (anti-HLA-DR), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF),cetuximab (anti-EGFR), gemtuzumab (anti-CD33), ibritumomab tiuxetan(anti-CD20); panitumumab (anti-EGFR); rituximab (anti-CD20); tositumomab(anti-CD20); GA101 (anti-CD20); and trastuzumab (anti-ErbB2). Suchantibodies are known in the art (e.g., U.S. Pat. Nos. 5,686,072;5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300;6,899,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785;7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; 7,585,491;7,612,180; 7,642,239; and U.S. Patent Application Publ. No. 20040202666(now abandoned); 20050271671; and 20060193865; the Examples section ofeach incorporated herein by reference.) Specific known antibodies of useinclude hPAM4 (U.S. Pat. No. 7,282,567), hA20 (U.S. Pat. No. 7,251,164),hA19 (U.S. Pat. No. 7,109,304), hIMMU31 (U.S. Pat. No. 7,300,655), hLL1(U.S. Pat. No. 7,312,318,), hLL2 (U.S. Pat. No. 7,074,403), hMu-9 (U.S.Pat. No. 7,387,773), hL243 (U.S. Pat. No. 7,612,180), hMN-14 (U.S. Pat.No. 6,676,924), hMN-15 (U.S. Pat. No. 7,541,440), hR1 (U.S. patentapplication Ser. No. 12/772,645), hRS7 (U.S. Pat. No. 7,238,785), hMN-3(U.S. Pat. No. 7,541,440), AB-PG1-XG1-026 (U.S. patent application Ser.No. 11/983,372, deposited as ATCC PTA-4405 and PTA-4406) and D2/B (WO2009/130575) the text of each recited patent or application isincorporated herein by reference with respect to the Figures andExamples sections.

Anti-TNF-α antibodies are known in the art and may be of use to treatimmune diseases, such as autoimmune disease, immune dysfunction (e.g.,graft-versus-host disease, organ transplant rejection) or diabetes.Known antibodies against TNF-α include the human antibody CDP571 (Ofeiet al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI,M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, Ill.);infliximab (Centocor, Malvern, Pa.); certolizumab pegol (UCB, Brussels,Belgium); and adalimumab (Abbott, Abbott Park, Ill.). These and manyother known anti-TNF-α antibodies may be used in the claimed methods andcompositions. Other antibodies of use for therapy of immunedysregulatory or autoimmune disease include, but are not limited to,anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab orhL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25);daclizumab (anti-CD25); efalizumab (anti-CD11a); muromonab-CD3 (anti-CD3receptor); anti-CD40L (UCB, Brussels, Belgium); natalizumab (anti-α4integrin) and omalizumab (anti-IgE).

Type-2 diabetes may be treated using known antibodies against B-cellantigens, such as CD22 (epratuzumab), CD74 (milatuzumab), CD19 (hA19),CD20 (veltuzumab) or HLA-DR (hL243) (see, e.g., Winer et al., 2011,Nature Med 17:610-18). Anti-CD3 antibodies also have been proposed fortherapy of type 1 diabetes (Cernea et al., 2010, Diabetes Metab Rev26:602-05).

Macrophage migration inhibitory factor (MIF) is an important regulatorof innate and adaptive immunity and apoptosis. It has been reported thatCD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med197:1467-76). The therapeutic effect of antagonistic anti-CD74antibodies on MIF-mediated intracellular pathways may be of use fortreatment of a broad range of disease states, such as cancers of thebladder, prostate, breast, lung, colon and chronic lymphocytic leukemia(e.g., Meyer-Siegler et al., 2004, BMC Cancer 12:34; Shachar & Haran,2011, Leuk Lymphoma 52:1446-54); autoimmune diseases such as rheumatoidarthritis and systemic lupus erythematosus (Morand & Leech, 2005, FrontBiosci 10:12-22; Shachar & Haran, 2011, Leuk Lymphoma 52:1446-54);kidney diseases such as renal allograft rejection (Lan, 2008, NephronExp Nephrol. 109:e79-83); and numerous inflammatory diseases(Meyer-Siegler et al., 2009, Mediators Inflamm epub Mar. 22, 2009;Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is anexemplary anti-CD74 antibody of therapeutic use for treatment ofMIF-mediated diseases.

Anti-CD3 antibodies have been reported to reduce development andprogression of atherosclerosis (Steffens et al., 2006, Circulation114:1977-84). Antibodies against oxidized LDL induced a regression ofestablished atherosclerosis in a mouse model (Ginsberg, 2007, J Am CollCardiol 52:2319-21). Anti-ICAM-1 antibody was shown to reduce ischemiccell damage after cerebral artery occlusion in rats (Zhang et al., 1994,Neurology 44:1747-51).

Antibody Allotypes

Immunogenicity of therapeutic antibodies is associated with increasedrisk of infusion reactions and decreased duration of therapeuticresponse (Baert et al., 2003, N Engl J Med 348:602-08). The extent towhich therapeutic antibodies induce an immune response in the host maybe determined in part by the allotype of the antibody (Stickler et al.,2011, Genes and Immunity 12:213-21). Antibody allotype is related toamino acid sequence variations at specific locations in the constantregion sequences of the antibody. The allotypes of IgG antibodiescontaining a heavy chain γ-type constant region are designated as Gmallotypes (1976, J Immunol 117:1056-59).

For the common IgG1 human antibodies, the most prevalent allotype isG1m1 (Stickler et al., 2011, Genes and Immunity 12:213-21). However, theG1m3 allotype also occurs frequently in Caucasians (Id.). It has beenreported that G1m1 antibodies contain allotypic sequences that tend toinduce an immune response when administered to non-G1m1 (nG1m1)recipients, such as G1m3 patients (Id.). Non-G1m1 allotype antibodiesare not as immunogenic when administered to G1m1 patients (Id.).

The human G1m1 allotype comprises the amino acids aspartic acid at Kabatposition 356 and leucine at Kabat position 358 in the CH3 sequence ofthe heavy chain IgG1. The nG1m1 allotype comprises the amino acidsglutamic acid at Kabat position 356 and methionine at Kabat position358. Both G1m1 and nG1m1 allotypes comprise a glutamic acid residue atKabat position 357 and the allotypes are sometimes referred to as DELand EEM allotypes. A non-limiting example of the heavy chain constantregion sequences for G1m1 and nG1m1 allotype antibodies is shown for theexemplary antibodies rituximab (SEQ ID NO:85) and veltuzumab (SEQ IDNO:86).

Rituximab heavy chain variable region sequence (SEQ ID NO: 85)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Veltuzumab heavy chain variable region(SEQ ID NO:86 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variationscharacteristic of IgG allotypes and their effect on immunogenicity. Theyreported that the G1m3 allotype is characterized by an arginine residueat Kabat position 214, compared to a lysine residue at Kabat 214 in theG1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acidat Kabat position 356, methionine at Kabat position 358 and alanine atKabat position 431. The G1m1,2 allotype was characterized by asparticacid at Kabat position 356, leucine at Kabat position 358 and glycine atKabat position 431. In addition to heavy chain constant region sequencevariants, Jefferis and Lefranc (2009) reported allotypic variants in thekappa light chain constant region, with the Km1 allotype characterizedby valine at Kabat position 153 and leucine at Kabat position 191, theKm1,2 allotype by alanine at Kabat position 153 and leucine at Kabatposition 191, and the Km3 allotype characterized by alanine at Kabatposition 153 and valine at Kabat position 191.

With regard to therapeutic antibodies, veltuzumab and rituximab are,respectively, humanized and chimeric IgG1 antibodies against CD20, ofuse for therapy of a wide variety of hematological malignancies and/orautoimmune diseases. Table 5 compares the allotype sequences ofrituximab vs. veltuzumab. As shown in Table 5, rituximab (G1m17,1) is aDEL allotype IgG1, with an additional sequence variation at Kabatposition 214 (heavy chain CH1) of lysine in rituximab vs. arginine inveltuzumab. It has been reported that veltuzumab is less immunogenic insubjects than rituximab (see, e.g., Morchhauser et al., 2009, J ClinOncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak &Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed tothe difference between humanized and chimeric antibodies. However, thedifference in allotypes between the EEM and DEL allotypes likely alsoaccounts for the lower immunogenicity of veltuzumab.

TABLE 5 Allotypes of Rituximab vs. Veltuzumab Heavy chain position andassociated allotypes Complete 214 356/358 431 allotype (allotype)(allotype) (allotype) Rituximab G1m17.1 K 17 D/L 1 A — Veltuzumab G1m3 R3 E/M — A —

In order to reduce the immunogenicity of therapeutic antibodies inindividuals of nG1m1 genotype, it is desirable to select the allotype ofthe antibody to correspond to the G1m3 allotype, characterized byarginine at Kabat 214, and the nG1m1,2 null-allotype, characterized byglutamic acid at Kabat position 356, methionine at Kabat position 358and alanine at Kabat position 431. Surprisingly, it was found thatrepeated subcutaneous administration of G1m3 antibodies over a longperiod of time did not result in a significant immune response. Inalternative embodiments, the human IgG4 heavy chain in common with theG1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356,methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicityappears to relate at least in part to the residues at those locations,use of the human IgG4 heavy chain constant region sequence fortherapeutic antibodies is also a preferred embodiment. Combinations ofG1m3 IgG1 antibodies with IgG4 antibodies may also be of use fortherapeutic administration.

Immunoconjugates

In certain embodiments, the antibodies or complexes may be conjugated toone or more therapeutic or diagnostic agents. The therapeutic agents donot need to be the same but can be different, e.g. a drug and aradioisotope. For example, ¹³¹I can be incorporated into a tyrosine ofan antibody or fusion protein and a drug attached to an epsilon aminogroup of a lysine residue. Therapeutic and diagnostic agents also can beattached, for example to reduced SH groups and/or to carbohydrate sidechains. Many methods for making covalent or non-covalent conjugates oftherapeutic or diagnostic agents with antibodies or fusion proteins areknown in the art and any such known method may be utilized.

A therapeutic or diagnostic agent can be attached at the hinge region ofa reduced antibody component via disulfide bond formation.Alternatively, such agents can be attached using a heterobifunctionalcross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP).Yu et al., Int. J. Cancer 56: 244 (1994). General techniques for suchconjugation are well-known in the art. See, for example, Wong, CHEMISTRYOF PROTEIN CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis etal., “Modification of Antibodies by Chemical Methods,” in MONOCLONALANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al. (eds.), pages187-230 (Wiley-Liss, Inc. 1995); Price, “Production and Characterizationof Synthetic Peptide-Derived Antibodies,” in MONOCLONAL ANTIBODIES:PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.),pages 60-84 (Cambridge University Press 1995). Alternatively, thetherapeutic or diagnostic agent can be conjugated via a carbohydratemoiety in the Fc region of the antibody. The carbohydrate group can beused to increase the loading of the same agent that is bound to a thiolgroup, or the carbohydrate moiety can be used to bind a differenttherapeutic or diagnostic agent.

Methods for conjugating peptides to antibody components via an antibodycarbohydrate moiety are well-known to those of skill in the art. See,for example, Shih et al., Int. J. Cancer 41: 832 (1988); Shih et al.,Int. J. Cancer 46: 1101 (1990); and Shih et al., U.S. Pat. No.5,057,313, incorporated herein in their entirety by reference. Thegeneral method involves reacting an antibody component having anoxidized carbohydrate portion with a carrier polymer that has at leastone free amine function. This reaction results in an initial Schiff base(imine) linkage, which can be stabilized by reduction to a secondaryamine to form the final conjugate.

The Fc region may be absent if the antibody used as the antibodycomponent is an antibody fragment. However, it is possible to introducea carbohydrate moiety into the light chain variable region of a fulllength antibody or antibody fragment. See, for example, Leung et al., J.Immunol. 154: 5919 (1995); Hansen et al., U.S. Pat. No. 5,443,953(1995), Leung et al., U.S. Pat. No. 6,254,868, incorporated herein byreference in their entirety. The engineered carbohydrate moiety is usedto attach the therapeutic or diagnostic agent.

In some embodiments, a chelating agent may be attached to an antibody,antibody fragment or fusion protein and used to chelate a therapeutic ordiagnostic agent, such as a radionuclide. Exemplary chelators includebut are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA.Methods of conjugation and use of chelating agents to attach metals orother ligands to proteins are well known in the art (see, e.g., U.S.Pat. No. 7,563,433, the Examples section of which is incorporated hereinby reference).

In certain embodiments, radioactive metals or paramagnetic ions may beattached to proteins or peptides by reaction with a reagent having along tail, to which may be attached a multiplicity of chelating groupsfor binding ions. Such a tail can be a polymer such as a polylysine,polysaccharide, or other derivatized or derivatizable chains havingpendant groups to which can be bound chelating groups such as, e.g.,ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaaceticacid (DTPA), porphyrins, polyamines, crown ethers,bis-thiosemicarbazones, polyoximes, and like groups known to be usefulfor this purpose.

Chelates may be directly linked to antibodies or peptides, for exampleas disclosed in U.S. Pat. No. 4,824,659, incorporated herein in itsentirety by reference. Particularly useful metal-chelate combinationsinclude 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, usedwith diagnostic isotopes in the general energy range of 60 to 4,000 keV,such as ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁶²Cu, ⁶⁴Cu, ¹⁸F, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga,^(99m)Tc, ^(94m)Tc, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br, for radioimaging. The samechelates, when complexed with non-radioactive metals, such as manganese,iron and gadolinium are useful for MRI. Macrocyclic chelates such asNOTA, DOTA, and TETA are of use with a variety of metals andradiometals, most particularly with radionuclides of gallium, yttriumand copper, respectively. Such metal-chelate complexes can be made verystable by tailoring the ring size to the metal of interest. Otherring-type chelates such as macrocyclic polyethers, which are of interestfor stably binding nuclides, such as ²²³Ra for RAIT are encompassed.

More recently, methods of ¹⁸F-labeling of use in PET scanning techniqueshave been disclosed, for example by reaction of F-18 with a metal orother atom, such as aluminum. The ¹⁸F—Al conjugate may be complexed withchelating groups, such as DOTA, NOTA or NETA that are attached directlyto antibodies or used to label targetable constructs in pre-targetingmethods. Such F-18 labeling techniques are disclosed in U.S. Pat. No.7,563,433, the Examples section of which is incorporated herein byreference.

Therapeutic Agents

In alternative embodiments, therapeutic agents such as cytotoxic agents,anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones,hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes orother agents may be used, either conjugated to the subject antibodycomplexes or separately administered before, simultaneously with, orafter the antibody complex. Drugs of use may possess a pharmaceuticalproperty selected from the group consisting of antimitotic, kinaseinhibitor, Bruton kinase inhibitor, alkylating, antimetabolite,antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents andcombinations thereof.

Exemplary drugs of use include, but are not limited to, 5-fluorouracil,afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib,AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib,bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin,10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin(CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin,cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine,dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin,daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX),cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicinglucuronide, erlotinib, estramustine, epidophyllotoxin, entinostat,estrogen receptor binding agents, etoposide (VP16), etoposideglucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine(FUdR), 3′,5′-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide,farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib,ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea,ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase,lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine,mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine,methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine,neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine,paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine,sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (anaqueous form of DTIC), transplatinum, thalidomide, thioguanine,thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine,vinblastine, vincristine, vinca alkaloids and ZD1839.

Toxins of use may include ricin, abrin, alpha toxin, saporin,ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcalenterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin,Pseudomonas exotoxin, and Pseudomonas endotoxin.

Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta andIP-10.

In certain embodiments, anti-angiogenic agents, such as angiostatin,baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PlGFpeptides and antibodies, anti-vascular growth factor antibodies,anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Krasantibodies, anti-cMET antibodies, anti-MIF (macrophagemigration-inhibitory factor) antibodies, laminin peptides, fibronectinpeptides, plasminogen activator inhibitors, tissue metalloproteinaseinhibitors, interferons, interleukin-12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin-2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470,endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine,bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

Immunomodulators of use may be selected from a cytokine, a stem cellgrowth factor, a lymphotoxin, a hematopoietic factor, a colonystimulating factor (CSF), an interferon (IFN), erythropoietin,thrombopoietin and a combination thereof. Specifically useful arelymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors,such as interleukin (IL), colony stimulating factor, such asgranulocyte-colony stimulating factor (G-CSF) or granulocytemacrophage-colony stimulating factor (GM-CSF), interferon, such asinterferons-α, -β or -γ, and stem cell growth factor, such as thatdesignated “S1 factor”. Included among the cytokines are growth hormonessuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; prostaglandin,fibroblast growth factor; prolactin; placental lactogen, OB protein;tumor necrosis factor-α and -β; mullerian-inhibiting substance; mousegonadotropin-associated peptide; inhibin; activin; vascular endothelialgrowth factor; integrin; thrombopoietin (TPO); nerve growth factors suchas NGF-β; platelet-growth factor; transforming growth factors (TGFs)such as TGF-α and TGF-β; insulin-like growth factor-I and -II;erythropoietin (EPO); osteoinductive factors; interferons such asinterferon-α, -β, and -γ; colony stimulating factors (CSFs) such asmacrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1α, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13,IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand orFLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factorand LT.

Radionuclides of use include, but are not limited to—¹¹¹In, ¹⁷⁷Lu,²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag,⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb,²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm,¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au, ²²⁷Th and ²¹¹Pb. The therapeuticradionuclide preferably has a decay-energy in the range of 20 to 6,000keV, preferably in the ranges 60 to 200 keV for an Auger emitter,100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alphaemitter. Maximum decay energies of useful beta-particle-emittingnuclides are preferably 20-5,000 keV, more preferably 100-4,000 keV, andmost preferably 500-2,500 keV. Also preferred are radionuclides thatsubstantially decay with Auger-emitting particles. For example, Co-58,Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161,Os-189m and Ir-192. Decay energies of useful beta-particle-emittingnuclides are preferably <1,000 keV, more preferably <100 keV, and mostpreferably <70 keV. Also preferred are radionuclides that substantiallydecay with generation of alpha-particles. Such radionuclides include,but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215,Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255. Decayenergies of useful alpha-particle-emitting radionuclides are preferably2,000-10,000 keV, more preferably 3,000-8,000 keV, and most preferably4,000-7,000 keV. Additional potential radioisotopes of use include ¹¹C,¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^(113m)In, ⁹⁵Ru, ⁹⁷Ru,¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^(121m)Te, ^(122m)Te, ^(125m)Te, ¹⁶⁵Tm,¹⁶⁷Tm, ¹⁶⁸Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rb, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au,⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb, and the like.Some useful diagnostic nuclides may include ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu,⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁸⁹Zr, ⁹⁴Tc, ^(94m)Tc, ^(99m)Tc, or ¹¹¹In. Radionuclidesand other metals may be delivered, for example, using chelating groupsattached to an antibody or conjugate. Macrocyclic chelates such as NOTA,DOTA, and TETA are of use with a variety of metals and radiometals, mostparticularly with radionuclides of gallium, yttrium and copper,respectively. Such metal-chelate complexes can be made very stable bytailoring the ring size to the metal of interest. Other ring-typechelates, such as macrocyclic polyethers for complexing ²²³Ra, may beused.

Therapeutic agents may include a photoactive agent or dye. Fluorescentcompositions, such as fluorochrome, and other chromogens, or dyes, suchas porphyrins sensitive to visible light, have been used to detect andto treat lesions by directing the suitable light to the lesion. Intherapy, this has been termed photoradiation, phototherapy, orphotodynamic therapy. See Joni et al. (eds.), PHOTODYNAMIC THERAPY OFTUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem.Britain (1986), 22:430. Moreover, monoclonal antibodies have beencoupled with photoactivated dyes for achieving phototherapy. See Mew etal., J. Immunol. (1983), 130:1473; idem., Cancer Res. (1985), 45:4380;Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol.Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422;Pelegrin et al., Cancer (1991), 67:2529.

Other useful therapeutic agents may comprise oligonucleotides,especially antisense oligonucleotides that preferably are directedagainst oncogenes and oncogene products, such as bcl-2 or p53. Apreferred form of therapeutic oligonucleotide is siRNA.

Diagnostic Agents

Diagnostic agents are preferably selected from the group consisting of aradionuclide, a radiological contrast agent, a paramagnetic ion, ametal, a fluorescent label, a chemiluminescent label, an ultrasoundcontrast agent and a photoactive agent. Such diagnostic agents are wellknown and any such known diagnostic agent may be used. Non-limitingexamples of diagnostic agents may include a radionuclide such as ¹¹⁰In,¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr,^(94m)Tc, ⁹⁴Tc, ^(99m)Tc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁵⁴⁻¹⁵⁸Gd, ³²P,¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^(52m)Mn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br,^(82m)Rb, ⁸³Sr, or other gamma-, beta-, or positron-emitters.Paramagnetic ions of use may include chromium (III), manganese (II),iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium(III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II),terbium (III), dysprosium (III), holmium (III) or erbium (III). Metalcontrast agents may include lanthanum (III), gold (III), lead (II) orbismuth (III). Ultrasound contrast agents may comprise liposomes, suchas gas filled liposomes. Radiopaque diagnostic agents may be selectedfrom compounds, barium compounds, gallium compounds, and thalliumcompounds. A wide variety of fluorescent labels are known in the art,including but not limited to fluorescein isothiocyanate, rhodamine,phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine. Chemiluminescent labels of use may include luminol,isoluminol, an aromatic acridinium ester, an imidazole, an acridiniumsalt or an oxalate ester.

Therapeutic Use

In another aspect, the invention relates to a method of treating asubject, comprising administering a therapeutically effective amount ofan antibody complex as described herein to a subject. Diseases that maybe treated with the antibody complexes described herein include, but arenot limited to immune diseases (e.g., SLE, RA, juvenile idiopathicarthritis, Crohn's disease, type 2 diabetes, Castleman's disease) orinflammatory diseases (e.g., sepsis, septic shock, inflammation,inflammatory bowel disease, inflammatory liver injury, acutepancreatitis). Such therapeutics can be given once or repeatedly,depending on the disease state and tolerability of the conjugate, andcan also be used optimally in combination with other therapeuticmodalities, such as immunomodulator therapy, immunotherapy,chemotherapy, antisense therapy, interference RNA therapy, gene therapy,and the like. Each combination will be adapted to patient condition andprior therapy, and other factors considered by the managing physician.

As used herein, the term “subject” refers to any animal (i.e.,vertebrates and invertebrates) including, but not limited to mammals,including humans. It is not intended that the term be limited to aparticular age or sex. Thus, adult and newborn subjects, as well asfetuses, whether male or female, are encompassed by the term.

In preferred embodiments, the antibodies that are used in the treatmentof human disease are human or humanized (CDR-grafted) versions ofantibodies; although murine and chimeric versions of antibodies can beused. Same species IgG molecules are mostly preferred to minimize immuneresponses. This is particularly important when considering repeattreatments. For humans, a human or humanized IgG antibody is less likelyto generate an anti-IgG immune response from patients.

In another preferred embodiment, diseases that may be treated using theantibody complexes include, but are not limited to immune dysregulationdisease and related autoimmune diseases, including Class III autoimmunediseases such as immune-mediated thrombocytopenias, such as acuteidiopathic thrombocytopenic purpura and chronic idiopathicthrombocytopenic purpura, dermatomyositis, Sjögren's syndrome, multiplesclerosis, Sydenham's chorea, myasthenia gravis, systemic lupuserythematosus, lupus nephritis, rheumatic fever, polyglandularsyndromes, bullous pemphigoid, diabetes mellitus, Henoch-Schonleinpurpura, post-streptococcal nephritis, erythema nodosum, Takayasu'sarteritis, Addison's disease, rheumatoid arthritis, sarcoidosis,ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritisnodosa, ankylosing spondylitis, Goodpasture's syndrome, thromboangitisobliterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto'sthyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis,rheumatoid arthritis, polymyositis/dermatomyositis, polychondritis,pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy,amyotrophic lateral sclerosis, tabes dorsalis, giant cellarteritis/polymyalgia, pernicious anemia, rapidly progressiveglomerulonephritis and fibrosing alveolitis, and also juvenile diabetes,as disclosed in U.S. Provisional Application Ser. No. 60/360,259, filedMar. 1, 2002 (now expired). Antibodies that may be of use forcombination therapy in these diseases include, but are not limited to,those reactive with HLA-DR antigens, B-cell and plasma-cell antigens(e.g., CD19, CD20, CD21, CD22, CD23, CD4, CD5, CD8, CD14, CD15, CD19,CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52,CD54, CD74, CD80, CD126, CD138, B7, MUC1, Ia, HM1.24, and HLA-DR), IL-6,IL-17. Since many of these autoimmune diseases are affected byautoantibodies made by aberrant B-cell populations, depletion of theseB-cells is a preferred method of autoimmune disease therapy. In apreferred embodiment, the anti-B-cell, anti-T-cell, or anti-macrophageor other such antibodies of use in the co-treatment of patients withautoimmune diseases also can be conjugated to result in more effectivetherapeutics to control the host responses involved in said autoimmunediseases, and can be given alone or in combination with othertherapeutic agents, such as TNF inhibitors or anti-IL-6R antibodies andthe like.

In a preferred embodiment, a more effective therapeutic agent can beprovided by using multivalent, multispecific antibodies. Exemplarybivalent and bispecific antibodies are found in U.S. Pat. Nos.7,387,772; 7,300,655; 7,238,785; and 7,282,567, the Examples section ofeach of which is incorporated herein by reference. These multivalent ormultispecific antibodies are particularly preferred in the targeting ofdisease associated cells which express multiple antigen targets and evenmultiple epitopes of the same antigen target, but which often evadeantibody targeting and sufficient binding for immunotherapy because ofinsufficient expression or availability of a single antigen target onthe cell. By targeting multiple antigens or epitopes, said antibodiesshow a higher binding and residence time on the target, thus affording ahigher saturation with the drug being targeted in this invention.

Formulation and Administration

Suitable routes of administration of the conjugates include, withoutlimitation, oral, parenteral, rectal, transmucosal, intestinaladministration, intramuscular, subcutaneous, intramedullary,intrathecal, direct intraventricular, intravenous, intravitreal,intraperitoneal, intranasal, or intraocular injections. The preferredroutes of administration are parenteral. Alternatively, one mayadminister the compound in a local rather than systemic manner, forexample, via injection of the compound directly into a solid tumor.

Antibody complexes or immunoconjugates can be formulated according toknown methods to prepare pharmaceutically useful compositions, wherebythe antibody complex or immunoconjugate is combined in a mixture with apharmaceutically suitable excipient. Sterile phosphate-buffered salineis one example of a pharmaceutically suitable excipient. Other suitableexcipients are well-known to those in the art. See, for example, Anselet al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5thEdition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON'SPHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990),and revised editions thereof.

The antibody complex or immunoconjugate can be formulated forintravenous administration via, for example, bolus injection orcontinuous infusion. Preferably, the antibody of the present inventionis infused over a period of less than about 4 hours, and morepreferably, over a period of less than about 3 hours. For example, thefirst 25-50 mg could be infused within 30 minutes, preferably even 15min, and the remainder infused over the next 2-3 hrs. Formulations forinjection can be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions cantake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and can contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient can be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

Additional pharmaceutical methods may be employed to control theduration of action of the antibody complex. Control release preparationscan be prepared through the use of polymers to complex or adsorb theantibody complex. For example, biocompatible polymers include matricesof poly(ethylene-co-vinyl acetate) and matrices of a polyanhydridecopolymer of a stearic acid dimer and sebacic acid. Sherwood et al.,Bio/Technology 10: 1446 (1992). The rate of release of an antibodycomplex or immunoconjugate from such a matrix depends upon the molecularweight, the amount of antibody complex or immunoconjugate within thematrix, and the size of dispersed particles. Saltzman et al., Biophys.J. 55: 163 (1989); Sherwood et al., supra. Other solid dosage forms aredescribed in Ansel et al., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERYSYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.),REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack PublishingCompany 1990), and revised editions thereof.

Generally, the dosage of an administered antibody complex orimmunoconjugate for humans will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition andprevious medical history. It may be desirable to provide the recipientwith a dosage that is in the range of from about 1 mg/kg to 25 mg/kg asa single intravenous infusion, although a lower or higher dosage alsomay be administered as circumstances dictate. A dosage of 1-20 mg/kg fora 70 kg patient, for example, is 70-1,400 mg, or 41-824 mg/m² for a1.7-m patient. The dosage may be repeated as needed, for example, onceper week for 4-10 weeks, once per week for 8 weeks, or once per week for4 weeks. It may also be given less frequently, such as every other weekfor several months, or monthly or quarterly for many months, as neededin a maintenance therapy.

Alternatively, an antibody complex or immunoconjugate may beadministered as one dosage every 2 or 3 weeks, repeated for a total ofat least 3 dosages. Or, twice per week for 4-6 weeks. If the dosage islowered to approximately 200-300 mg/m² (340 mg per dosage for a 1.7-mpatient, or 4.9 mg/kg for a 70 kg patient), it may be administered onceor even twice weekly for 4 to 10 weeks. Alternatively, the dosageschedule may be decreased, namely every 2 or 3 weeks for 2-3 months. Ithas been determined, however, that even higher doses, such as 20 mg/kgonce weekly or once every 2-3 weeks can be administered by slow i.v.infusion, for repeated dosing cycles. The dosing schedule can optionallybe repeated at other intervals and dosage may be given through variousparenteral routes, with appropriate adjustment of the dose and schedule.

Expression Vectors

Still other embodiments may concern DNA sequences comprising a nucleicacid encoding an antibody, antibody fragment, toxin or constituentfusion protein of an antibody complex, such as a DNL® construct. Fusionproteins may comprise an antibody or fragment or toxin attached to, forexample, an AD or DDD moiety.

Various embodiments relate to expression vectors comprising the codingDNA sequences. The vectors may contain sequences encoding the light andheavy chain constant regions and the hinge region of a humanimmunoglobulin to which may be attached chimeric, humanized or humanvariable region sequences. The vectors may additionally containpromoters that express the encoded protein(s) in a selected host cell,enhancers and signal or leader sequences. Vectors that are particularlyuseful are pdHL2 or GS. More preferably, the light and heavy chainconstant regions and hinge region may be from a human EU myelomaimmunoglobulin, where optionally at least one of the amino acid in theallotype positions is changed to that found in a different IgG1allotype, and wherein optionally amino acid 253 of the heavy chain of EUbased on the EU number system may be replaced with alanine. See Edelmanet al., Proc. Natl. Acad. Sci USA 63: 78-85 (1969). In otherembodiments, an IgG1 sequence may be converted to an IgG4 sequence.

The skilled artisan will realize that methods of genetically engineeringexpression constructs and insertion into host cells to expressengineered proteins are well known in the art and a matter of routineexperimentation. Host cells and methods of expression of clonedantibodies or fragments have been described, for example, in U.S. Pat.Nos. 7,531,327 and 7,537,930, the Examples section of each incorporatedherein by reference.

Kits

Various embodiments may concern kits containing components suitable fortreating or diagnosing diseased tissue in a patient. Exemplary kits maycontain one or more antibody complexes as described herein. If thecomposition containing components for administration is not formulatedfor delivery via the alimentary canal, such as by oral delivery, adevice capable of delivering the kit components through some other routemay be included. One type of device, for applications such as parenteraldelivery, is a syringe that is used to inject the composition into thebody of a subject. Inhalation devices may also be used. In certainembodiments, a therapeutic agent may be provided in the form of aprefilled syringe or autoinjection pen containing a sterile, liquidformulation or lyophilized preparation.

The kit components may be packaged together or separated into two ormore containers. In some embodiments, the containers may be vials thatcontain sterile, lyophilized formulations of a composition that aresuitable for reconstitution. A kit may also contain one or more bufferssuitable for reconstitution and/or dilution of other reagents. Othercontainers that may be used include, but are not limited to, a pouch,tray, box, tube, or the like. Kit components may be packaged andmaintained sterilely within the containers. Another component that canbe included is instructions to a person using a kit for its use.

EXAMPLES

In the working Examples below, the DOCK-AND-LOCK® (DNL®) technology wasused to generate the first bispecific antibody (bsAb) with high potencyto neutralize both TNF-α and IL-6. This prototype DNL® construct,designated cT*-(c6)-(c6), comprises a chimeric anti-TNF-α IgG linked atthe carboxyl terminus of each light chain to a pair of dimerized Fab'sderived from a chimeric anti-IL-6 antibody, thus featuring a hexavalentbsAb capable of blocking 2 and 4 molecules of TNF-α and IL-6,respectively, as well as a fully functional Fc. As discussed below, theexemplary anti-TNF-α/anti-IL-6 bispecific antibody showed potentactivity in in vitro assays designed to test efficacy for immunediseases such as SLE or RA. However, the person of ordinary skill willrealize that the subject complexes of use are not limited to thespecific DNL® cT*-(c6)-(c6) complex discussed below, but more generallyencompass bispecific antibodies and/or antigen-binding antibodyfragments with at least one binding site for IL-6 an at least onebinding site for TNF-α.

Example 1. Generation of Neutralizing Mouse Anti-Human IL-6 MonoclonalAntibody

The 2-3B2 mouse monoclonal antibody against human IL-6 was producedusing standard immunological techniques, discussed below, that may beused to make anti-human IL-6 antibodies in general.

Recombinant human IL-6 (rhIL-6) was obtained from ProSpec-TanyTechnoGene Ltd. (Rehovot, Israel). Multiple mice were initiallyimmunized with 30 μg rhIL-6 administered i.p., followed by boosterinjections of 30 or 10 μg with or without adjuvant, according to astandard boosting schedule. Animals were tested for presence ofanti-IL-6 antibodies by ELISA assay using rhIL-6 coated microtiterplates and serial dilutions of serum. Prior to fusion, the presence ofneutralizing anti-IL-6 antibodies was detected by the ability to blockIL-6 stimulated protein phosphorylation (of STAT3) using Westernblotting (data not shown).

Cells secreting neutralizing anti-IL-6 antibodies were fused with theP3-X63.Ag8.653 myeloma cell line by PEG mediated cell fusion usingstandard techniques to generate antibody-secreting hybridomas celllines. The 2-3B2, 4-4F5 and 4-4E6 anti-IL-6 clones were obtained byselection on HAT medium, cloning and subcloning. Supernatants fromisolated clones containing neutralizing anti-IL-6 were detected by theability to block IL-6 stimulated STAT3 protein phosphorylation,determined by Western blotting (FIG. 1). Clones 2-3B2, 4-4E6 and controlanti-IL-6 MAb206, but not clone 4-4F5, were able to block rhIL-6 inducedphosphorylation (FIG. 1). Size exclusion HPLC of antibodies purified byprotein A column chromatography demonstrated the presence of homogeneousantibodies, which was confirmed by SDS-PAGE (data not shown). Isotypingusing an SBA CLONETYPING™ system showed that the anti-IL-6 antibodieswere IgG1/κ murine isotypes.

Binding to human IL-6 was determined Western blotting against rhIL-6(FIG. 2). Based on the intensity of labeling using identicalconcentrations of antibody, it was determined that the 2-3B2 clone (FIG.2A) showed higher affinity for human IL-6 than the 4-4E6 clone (FIG. 2B)and 2-3B2 was selected for production of chimeric and humanizedanti-IL-6 antibodies. Serial dilution demonstrated that the 2-3B2antibody was about 100-fold more potent than 4-4E6 for inhibiting theIL-6 induced phosphorylation of STAT3 (FIG. 2A-B). Neither antibodybound to murine IL-6 (not shown).

Example 2. Generation of Neutralizing Mouse Anti-Human TNF-α MonoclonalAntibody

Monoclonal antibodies against human TNF-α were prepared using standardtechniques, as discussed in Example 1 above for IL-6. Mice wereimmunized with recombinant human TNF-α obtained from ProSpec-TanyTechnoGene Ltd. (Rehovot, Israel). Testing of serum from immunized micefor anti-TNF-α antibodies was performed by ELISA (data not shown).

Neutralizing antibodies were also detected by cytotoxicity assay.Briefly, WEHI 164 cells (mouse fibrosarcoma) were cultured in RPMIcomplete media. Cells were plated at a density of 1×10⁴ cells/well in 75μL of medium in 96-well plates and kept in a 37° C. incubator overnightbefore the assay. On the day of the assay, sera from the immunized micewere diluted 1:25, 1:125, 1:625, 1:3,125, 1:15,625, and 1:78,125 in RPMIcomplete medium containing 8 μg/mL of actinomycin-D and 0.4 ng/mL ofrhTNF-α. Twenty-five μL of the diluted sera were added to the cells inthe corresponding wells. The addition of the sera to the cells made thefinal dilutions of the sera as 1:100, 1:500, 1:2,500, 1:12,500,1:62,500, and 1:312,500. The final concentration of actinomycin-D was 2μg/mL and rhTNF-α was 0.1 ng/mL. Plates were incubated in a 37° C./5%CO2 incubator for 20 hours. After this incubation, 20 μL of MTS reagentwas added to all the wells and the absorbance in each well determined ina plate reader at 490 nm after two hours. As a negative control, serumfrom a naive mouse (not immunized) was diluted in a like manner. One setof wells was incubated with only actinomycin-D and rhTNF-α to determinemaximum growth inhibition. Another set of cells remained untreated(cells grown in media lacking actinomycin-D and rhTNF-α). Growthinhibition was measured as percent of untreated control cell growth.

The results of the cytotoxicity assay are shown in FIG. 3. Serum fromeach of the inoculated mice showed the ability to neutralize rhTNF-αmediated cytotoxicity. Serum from mouse #3 showed the greatest abilityto inhibit rhTNF-α mediated cytotoxicity.

Hybridomas were produced from splenocytes of mice showing the presenceof anti-TNF-α antibodies by PEG fusion, essentially as discussed above.Selection of fused hybridomas was performed using HAT medium.Neutralizing clones 4C9 and 4D3 were obtained from mouse #3. Afterfurther subcloning, antibodies were purified by chromatography onprotein G columns. Purified antibodies were determined to be homogeneousby size separation HPLC and SDS-PAGE (data not shown). Isotype analysis,performed as discussed above, showed that 4C9 was IgG1/κ while 4D3 wasIgG2a/κ.

The ability of anti-TNF-α antibodies from clones 4C9D11 and 4D3B11 toneutralize TNF-α-mediated cytotoxicity was determined (FIG. 4). WEHI 164cells were seeded at 1×10⁴ cells/well into 96-well plates and grown in200 μL of RPMI complete medium overnight. On the day of the assay,supernatants from clones were collected and diluted 1:2. A further 1:5dilution was made thereafter. Each dilution was made in RPMI completemedium containing a final concentration of actinomycin-D at 2 μg/mL andrhTNF-α at 0.1 ng/mL. Before addition of the diluted supernatant, themedium in the plate for WEHI 164 cells growth was removed, and replacedwith the diluted supernatant in the corresponding wells, 100 μL/well.The plate was incubated for 20 hours at 37° C. in a 5% CO2 incubator.After this incubation, 20 μL of MTS was added to all the wells and theabsorbance in each well determined in a plate reader at 490 nm after twohours. As a negative control, supernatant from a clone which stoppedproducing antibody (ELISA negative) was diluted in a like manner. Oneset of wells was incubated with only actinomycin-D and rhTNF-α todetermine maximum growth inhibition. Another set of cells remaineduntreated (cells grown in media lacking actinomycin-D and rhTNF-α).Growth inhibition was measured as percent of untreated control cellgrowth. The antibody from clone 4D3B11 was more effective at blockingTNF-α mediated cytotoxicity in this assay (FIG. 4).

Antibody binding specificity was determined by Western blotting againstrhTNF-α. Under reducing conditions, 4D3B11C4 and the anti-TNF-α antibodyREMICADE® (infliximab) showed no or weak binding to human TNF-α, with nobinding to human TNF-β or murine TNF-α (not shown). Under the samereducing conditions, antibody 4C9D11G11 showed strong binding to humanTNF-α, with no binding to human TNF-β or murine TNF-α (not shown).

Neutralization of rhTNF-α induced cytotoxicity by anti-TNF-α antibodieswas determined in a different in vitro system (FIG. 5). L929 cells(mouse fibroblasts) were seeded at 2×10⁴ cells/well into 96-well platesand grown in 90 μL of MEM medium overnight (10% horse serum completemedium). On the following day, the purified antibodies were diluted 1:5in MEM medium (containing a final concentration of actinomycin-D at 20μg/mL and rhTNF-α at 1 ng/mL) for an antibody concentration range of10,000 to 3.2 ng/mL. The antibodies were pre-incubated with rhTNF-α atRT for one hour. After this pre-incubation, 10 μL of the dilutedantibodies were then added to the 90 μL cells in the correspondingwells, that made the final concentration of the antibodies from 1000 to0.32 ng/mL, with a final concentration of actinomycin-D and rhTNF-α at 2μg/mL and 0.1 ng/mL, respectively. The plate was incubated for 20 hoursat 37° C. Following this incubation, 20 μL of MTS was added to all thewells and the absorbance in each well determined in a plate reader at490 nm after two hours. As a negative control, an anti-hTNF-α antibody,4C3 (non-neutralizing), was diluted in a like manner. REMICADE®, thecommercial anti-TNF-α antibody was also diluted in a like manner as apositive control. One set of wells was incubated with only actinomycin-Dand rhTNF-α to determine maximum growth inhibition.

Under these conditions, the 4C9D11G11 antibody (EC₅₀ 11.2 ng/mL) wasmore effective than 4D3B11C4 (EC₅₀ 22.1 ng/mL) at inhibitingTNF-α-induced cytotoxicity (FIG. 5). Neither monoclonal antibody was aseffective as REMICADE® (EC₅₀ 3.6 ng/mL) (FIG. 5).

An assay was performed for antibody based neutralization ofrhTNF-α-induced cell surface expression of ICAM-1 (FIG. 6). ECV-304cells (a derivative of T24, bladder cancer cell line) were seeded at2×10⁵ cells/well into 6-well plates, grown in 10% FBS Medium 199 for 6hours for attaching. Varying doses of the mAbs or REMICADE® (positivecontrol) were mixed with constant amounts of rhTNF-α (10 ng/mL). Themixture of the antibodies and rhTNF-α was pre-incubated at 37° C. fortwo hours, and then pipetted into the appropriate corresponding wells induplicate. Cells were then grown for 72 hours in a 37° C. incubator.After this incubation, supernatant was removed and cells weretrypsinized and transferred to 15 mL tubes. Cells were washed with coldPBS/0.5% BSA two times, supernatant was removed and the cell pelletswere re-suspended in the residual wash buffer (˜100 μL). An aliquot of25 μL from the cell suspension from each sample was then transferred to4 mL flow tubes. Cells were Fc-blocked by treatment with 1 μg of humanIgG for 15 min at RT and then incubated with fluorescent-conjugatedanti-CD54 reagent for 45 min at 4° C. Cells were then washed with 4 mLof PBS/5% BSA for two times and re-suspended in 400 μL of PBS and thensubjected to flow-cytometric analysis (FACS). One set of cells remaineduntreated as background fluorescent control. Another set of cellstreated with only 10 ng/mL of rhTNF-α served as the positive control forobtaining maximum fluorescent (i.e. maximum ICAM-1 up-regulation).

The 4C9 clone again showed higher neutralizing activity than the 4D3clone and 4C9 was selected for chimerization.

Example 3. Production of Chimeric Anti-IL-6 Antibody from 2-3B2Hybridoma

Total RNA was extracted from hybridomas 2-3B2 cells by standardtechniques and mRNA was separated from the total RNA fraction. The mRNAwas used as a template for VH and VK cDNA synthesis, using a QIAGEN®OneStep RT-PCR kit. Primers used were as shown below (restriction sitesare underlined).

Vk1 BACK (PNAS 86:3833-3837,1989) (SEQ ID NO: 90)GACATTCAGCTGACCCAGTCTCCA CK3′-BH: (Biotechniques 15:286-291, 1993)(SEQ ID NO: 91) GCCGGATCCTCACTGGATGGTGGGAAGATGGATACAVH1 BACK: (PNAS 86:3833-3837, 1989)AGGTSMARCTGCAGSAGTCWGG (SEQ ID NO: 92, S = C/G, M = A/C, R = A/G, W =A/T) CH1-C: (Clinical Cancer Res 5:3095s-3100s, 1999) (SEQ ID NO: 93)AGCTGGGAAGGTGTGCAC

The VH and Vk cDNA sequences were cloned into the pGEMT vector forsequencing by the Sanger dideoxy technique, using an automated DNAsequencer. The putative VH (SEQ ID NO:94) and Vk (SEQ ID NO:96) murineamino sequences are shown in FIG. 7 and FIG. 8. The locations of theheavy and light chain CDR sequences are proposed, based on homology withthe known heavy and light chain antibody sequences of B34871 (SEQ IDNO:95) and AAB53778.1 (SEQ ID NO:97), respectively, from the NCBIprotein sequence database. The indicated 2-3B2 heavy chain CDR sequencesare CDR1 (GFTFSRFGMH, SEQ ID NO:107), CDR2 (YIGRGSSTIYYADTVKG, SEQ IDNO:108) and CDR3 (SNWDGAMDY, SEQ ID NO:109). The 2-3B2 light chain CDRsequences are CDR1 (RASGNIHNFLA, SEQ ID NO:110), CDR2 (NAETLAD, SEQ IDNO:111) and CDR3 (QHFWSTPWT, SEQ ID NO:112).

The VH and VK sequences from the 2-3B2 anti-IL-6 antibody and the VH andVK sequences from the 4C9 anti-TNF-α antibody were used to make acIL6/TNFα DVD (dual variable domain) antibody construct. (See, e.g., Wuet al., 2009, MAbs 1:339-47.) The resulting bispecific DVD construct wascompared with the parent 2-3B2 anti-IL-6 antibody for the ability toinhibit IL-6 induced phosphorylation of STAT3 on HT-29 cells (FIG. 9).As shown in FIG. 9, the DVD construct showed the same efficacy as theparent anti-IL-6 antibody for inhibition of IL-6 mediatedphosphorylation.

The sequences for restriction sites and leader peptides for cloning intovector pdHL2 were added to the VH and VK sequences of 2-3B2. Thecomplete sequences were synthesized commercially (GenScript, Piscataway,N.J.). The 2-3B2-VH-pUC57 and 2-3B2-VK-pUC57 vectors were produced byincorporating the VH sequence as a XhoI-HindIII insert and the VKsequence as a XbaI-BamH1 insert into corresponding sites in pUC57. Avector expressing chimeric 2-3B2 antibody was produced starting with thehA20-pdHL2-IgG vector (see, e.g., Goldenberg et al., 2002, Blood 100:11Abstract 2260). The hA20-VH sequence was replaced with cIL6-VH and thehA20-VK sequence was replaced with cIL6-VK by restriction enzymedigestion and ligation. The resulting chimeric 2-3B2 antibody comprisedthe murine VH and VK sequences of 2-3B2, attached to human antibodyconstant region sequences. After transfection, screening and antibodypurification on a protein A column, a chimeric anti-IL6 clone 1B5(c-IL6-1B5) was obtained as a homogeneous antibody preparation, asconfirmed by HPLC and SDS-PAGE (not shown). The final clone isidentified as 1B5A9.

Example 4. Production of Chimeric Anti-TNF-α Antibody from 4C9 Hybridoma

Total RNA was extracted from hybridomas 4C9 cells by standard techniquesand mRNA was separated from the total RNA fraction. The mRNA was used asa template for VH and VK cDNA synthesis, using a PHUSION® High FidelityPCR kit (Thermo Scientific, Pittsburgh, Pa.). Primers used were asdisclosed in Example 3 above.

The VH and Vk cDNA sequences were cloned into the pGEMT vector forsequencing by the Sanger dideoxy technique, using an automated DNAsequencer. The putative VH (SEQ ID NO:98) and Vk (SEQ ID NO:100) murineamino sequences are shown in FIG. 10 and FIG. 11. The locations of theheavy and light chain CDR sequences are proposed, based on homology withthe known heavy and light chain antibody sequences of AAS66033.1 (SEQ IDNO:99) and AAS66032.1 (SEQ ID NO:101), respectively, from the NCBIprotein sequence database. The indicated 4C9 heavy chain CDR sequencesare CDR1 (GFWN, SEQ ID NO:113), CDR2 (YISYSGRTYYNPSLKS, SEQ ID NO:114)and CDR3 (DANYVLDY, SEQ ID NO:115). The 4C9 light chain CDR sequencesare CDR1 (KSSQSLLNSSTQKNYLA, SEQ ID NO:116), CDR2 (FASARES, SEQ IDNO:117) and CDR3 (QQHYRTPFT, SEQ ID NO:118).

An optimized DNA sequence encoding the TNF-α VH, also comprising a 5′leader sequence and 3′ flanking sequence, was designed as shown in SEQID NO:102 below and cloned into pdHL2. The optimized 4C9-VH sequence isunderlined. The DNA sequence was synthesized by GenScript (Piscataway,N.J.).

(SEQ ID NO: 102) CTCGAGCACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCGTGCAGCTGCAGGAGAGCGGACCCTCCCTGGTGAAGCCTAGTCAGACCCTGAGCCTGACATGCTCCGTGACTGGGGACTCTATCACCAGTGGCTTCTGGAACTGGATTCGGAAGTTCCCAGGAAACAAGTTTGAATACATGGGATATATCTCTTACAGTGGGCGCACATACTATAACCCCAGCCTGAAGTCCAGGCTGTCTATTACAAGAGACACTTCTAAAAACCAGTTTTATCTGCAGCTGAACAGCGTGACTGCCGAGGATACTGCTACCTACTATTGTGCCAGGGACGCTAATTATGTGCTGGATTACTGGGGCCAGGGAACCACACTGACCGTGAGCTCCGGTGAGTCCTTACAACCTCTCTCTTCTATTCAGCTTAAATAGATTTTACTGCATTTGTTGGGGGGGAAATGTGTGTATCTGAATTTCAGGTCATGAAGGACTAGGGACACCTTGGGAGTCAGAAAGGGTCATTGGGAAGCTT 

An optimized DNA sequence encoding the TNF-α VK, also comprising a 5′leader sequence and 3′ flanking sequence, was designed as shown in SEQID NO:103 below and cloned into pdHL2. The optimized 4C9-VK sequence isunderlined. The DNA sequence was synthesized by GenScript (Piscataway,N.J.).

(SEQ ID NO: 103) TCTAGACACAGGACCTCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTAAGGGGCTCACAGTAGCAGGCTTGAGGTCTGGACATATATATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCGACATCCAGCTGACCCAGAGCCCCAGCTCCCTGGCTATGTCCGTGGGACAGAAGGTGACAATGAACTGCAAATCTAGTCAGTCTCTGCTGAACAGCTCCACTCAGAAGAATTACCTGGCTTGGTTCCAGCAGAAGCCCGGGCAGAGTCCTAAACTGCTGGTGTATTTTGCCTCTGCTAGGGAGAGTGGCGTGCCAGACAGATTCATCGGCAGCGGCAGCGGGACCGATTTTACCCTGACAATTTCTAGTGTGCAGGCCGAGGACCTGGCTGATTACTTCTGTCAGCAGCACTATCGGACTCCCTTCACCTTTGGCTCCGGAACAAAGCTGGAGATCAAGCGTGAGTAGAATTTAAACTTTGCTTCCTCAGTTGGATCC 

The VH and VK coding sequences were inserted into pUC57 and then pdHL2for expression of the chimeric 4C9 antibody as discussed in Example 3above. The chimeric 4C9 was produced by transfection of pdHL2, screeningfor transfectants and antibody purification on a protein A column. Theselected clone was designated 6A9. The purified antibody was determinedto be homogeneous by HPLC and SDS-PAGE (not shown). A binding affinityassay for chimeric anti-TNF-α showed a dissociation constant (K_(D)) of4.13 e⁻¹¹ (not shown).

Example 5. Construction of CH1-DDD2-cFab-Anti-IL-6-pGSHL

The hLL2-Fab-DDD2-pGSHL#2 plasmid (see, e.g., WO2013181087A2; Rossi etal., 2009, Blood 113:6161-71; U.S. Patent Publ. Nos. 20130323204,20140212425) was used as a starting material for production of a DDD2conjugated Fab anti-IL-6 antibody fragment. The hLL2-DDD2 plasmid wasdigested with XbaI/XhoI and the 6577 bp vector was isolated. cIL6-pdHL2(Example 3) was digested with XbaI/XhoI and the 2604 bp cIL6 codinginsert was isolated. The two were ligated to form the 9182 bpVk-cIL6-Fab-DDD2-pGSHL vector. After screening by PstI digestion andelectrophoresis, the 9182 bp vector was digested withXhoI/Hind3/Alkaline phosphatase and an 8536 vector was isolated. ThecIL6-pdHL2 vector, comprising a 648 bp cIL6-VH coding insert wasdigested with XhoI/Hind3. The 648 bp VH encoding insert was ligated withthe 8536 bp vector and VK insert to generateC_(H)1-DDD2-cFab-anti-IL-6-pGSHL. The final construct was thentransfected, clones were picked and purified by Kappa-select (GEHealthcare Life Sciences, Piscataway, N.J.). The purified antibodyproduct of C_(H)1-DDD2-cFab-anti-IL-6 appeared homogeneous on HPLC andSDS-PAGE (not shown). The DDD2-derivatized cIL6-Fab showed equivalentactivity to the underivatized cIL6 or an hR1-(IL6)₄ construct whenassayed for inhibition of IL-6 induced STAT3 phosphorylation (notshown).

Example 6. Construction of C_(K)-AD2-cIgG-Anti-TNF-α-pdHL2

The Ck-AD2-IgG-hA20-pdHL2 plasmid (see, e.g., WO201262583A1; Chang etal., 2012, PLoS ONE 7(8): e44235; U.S. Patent Publ. Nos. 20130323204,20070140966) was used as a starting material for production of an AD2conjugated IgG anti-TNF-α antibody. The Ck-AD2-hA20 plasmid was digestedwith BamHI/XhoI to obtain the Ck-AD2 coding portion. PlasmidcIgG-anti-TNF-α-pdHL2 (Example 4) was digested with BamHI/XhoI to obtainΔC_(K)-cIgG-anti-TNF-α-pdHL2. The two were ligated to formCk-AD2-cIgG-anti-TNF-α-pdHL2 (see FIG. 12). TheC_(K)-AD2-cIgG-anti-TNF-α-pdHL2 vector was used to transform DHFαcompetent cells. Colonies were picked and purified by mini-Prep. PlasmidDNA was analyzed by restriction endonuclease digestion and agarose gelelectrophoresis (not shown). The plasmid DNA was purified by Maxi-Prepand the insert was DNA sequenced. The DNA sequences encoding cTNF-α-VH,AD2 and cTNF-α-VK are shown in SEQ ID NOs 104-106 below.

cTNF-α-VH (SEQ ID NO: 104)GTGCAGCTGCAGGAGAGCGGACCCTCCCTGGTGAAGCCTAGTCAGACCCTGAGCCTGACATGCTCCGTGACTGGGGACTCTATCACCAGTGGCTTCTGGAACTGGATTCGGAAGTTCCCAGGAAACAAGTTTGAATACATGGGATATATCTCTTACAGTGGGCGCACATACTATAACCCCAGCCTGAAGTCCAGGCTGTCTATTACAAGAGACACTTCTAAAAACCAGTTTTATCTGCAGCTGAACAGCGTGACTGCCGAGGATACTGCTACCTACTATTGTGCCAGGGACGCTAATTATGTGCTGGATTACTGGGGCCAGGGAACCACACTGACCGTGAGCTCC  AD2 (SEQ ID NO: 105)TGTGGCCAGATCGAGTACCTGGCCAAGCAGATCGTGGACAACGCCATCCA GCAGGCCGGGTGC cTNF-α-VK (SEQ ID NO: 106)GACATCCAGCTGACCCAGAGCCCCAGCTCCCTGGCTATGTCCGTGGGACAGAAGGTGACAATGAACTGCAAATCTAGTCAGTCTCTGCTGAACAGCTCCACTCAGAAGAATTACCTGGCTTGGTTCCAGCAGAAGCCCGGGCAGAGTCCTAAACTGCTGGTGTATTTTGCCTCTGCTAGGGAGAGTGGCGTGCCAGACAGATTCATCGGCAGCGGCAGCGGGACCGATTTTACCCTGACAATTTCTAGTGTGCAGGCCGAGGACCTGGCTGATTACTTCTGTCAGCAGCACTATCGGACTCCCTTCACCTTTGGCTCCGGAACAAAGCTGGAGATCAAGCGTGAGTAGAA TTTAAACTTTGCT

After transfection, screening, expression and antibody purification,clone 4A5 encoding C_(K)-AD2-cIgG-4A5 was obtained.

Example 7. Construction of cT*-(c6)-(c6) Anti-IL-6/Anti-TNF-α BispecificDNL® Complex

The Ck-AD2-cIgG-4A5 and C_(H)1-DDD2-cFab-anti-IL-6 fusion proteins wereused to make a DOCK-AND-LOCK® (DNL)® complex, using techniques disclosedherein and in issued U.S. Pat. Nos. 7,550,143; 7,521,056; 7,534,866;7,527,787; 7,666,400; 7,858,070; 7,871,622; 7,906,121; 7,906,118;8,163,291; 7,901,680; 7,981,398; 8,003,111; 8,034,352; 8,562,988;8,211,440; 8,491,914; 8,282,934; 8,246,960; 8,349,332; 8,277,817;8,158,129; 8,475,794; 8,597,659; 8,481,041; 8,435,540 and 8,551,480, theExamples section of each incorporated herein by reference.

The intact DNL® complex was formed by mixing the AD2 and DDD2 componentstogether under reducing conditions and allowing the complementarysequences on the DDD moiety to form a dimer that binds to the AD moiety.Twenty five mg of C_(K)-AD2-cIgG-4A5 was mixed with 50 mg ofC_(H)1-DDD2-cFab-anti-IL-6. A 1/10 volume of 1 M Tris, pH 7.5, 1 mMEDTA, 2 mM reduced glutathione was added to the reaction and theproteins were reduced overnight at room temperature. The complexes werethen oxidized with 4 mM oxidized glutathione at room temperature for 3hours to form disulfide bonds between the AD2 and DDD2 moieties tostabilize the complex.

Chromatography of the complex on a MAB SELECT™ column was performed.After loading, the column was washed with 0.04 M PBS, pH 7.4+1 mMoxidized glutathione, followed by a PBS wash and elution with 0.1 Mcitrate (pH 3.5). The elution volume was 25 ml (2.5 ml of 3 M Tris, pH8.6+22.5 ml of eluate). The concentration measured by OD280 was 2.3mg/ml (57.5 mg total).

The product was dialyzed against two 5-L changes of 0.04 M PBS, pH 7.4.The final concentration by OD280 was 1.8 mg/ml (52.5 mg total). Purifiedcomplex was analyzed by SE-HPLC, which confirmed the presence ofcT*-(c6)-(c6) as an apparently homogeneous peak (not shown). The resultswere confirmed by SDS-PAGE. The activity of the purified cT*-(c6)-(c6)bispecific antibody complex was then examined.

The cT*-(c6)-(c6) complex showed greater activity than theFab-DDD2-cIL-6 protein for inhibiting IL-6 induced phosphorylation ofSTAT3 (FIG. 13). HT-29 cells were seeded at 2×10⁶ cells/well in 6-wellplates, grown overnight. The indicated antibodies were pre-incubatedwith hIL6 at 37° C. for 1 hour. Then media containing rhIL-6 alone or incombination with antibodies was added to the HT-29 cells for 30 min at37° C. After the incubation, the supernatant was removed, cells werewashed and lysed.

Two SDS-PAGE gels were run, transferred to nitrocellulose membranes.Membranes were cut at 60 KDa, the upper portions was probed with eitheranti-p-STAT3 or anti-t-STAT3 (FIG. 13). The lower portions were probedwith b-actin for loading control (FIG. 13). This assay showed thatTNF-(IL6)-(IL6) neutralized IL-6 with similar or greater potencycompared to anti-IL-6 Fab-DDD2.

FIG. 14 shows that the cT*-(c6)-(c6) complex was able to neutralizenatural IL-6 induced phosphorylation of STAT3 in HT-29 cells. HT-29cells were seeded at 2×10⁶ cells/well in 6-well plates and grownovernight. TNF-(IL6)-(IL6) or chimeric anti-IL6 1B5A9 was pre-incubatedwith the supernatant containing 10 ng/mL of natural IL6 released fromcollagen Type II stimulated RA patient PBMCs at 37° C. for 1 hour. Atthe end of the incubation, the supernatant containing natural IL-6 aloneor in combination with antibodies was added to the HT-29 cells for 30min at 37° C. After incubation, the supernatant was removed and cellswere washed and lysed. Two SDS-PAGE gels were run, transferred tonitrocellulose membranes. Membranes were cut at 60 KDa, the upperportion was probed with either anti-p-STAT3 or anti-t-STAT3 (FIG. 14).The lower portions were probed with b-actin for loading control (FIG.14).

The ability to neutralize TNF-α induced cell death was also examined forcT*-(c6)-(c6) compared to other anti-TNF-α antibody constructs (FIG.15). In the presence of 2 μg/mL of actinomycin-D, recombinant humanTNF-α at 0.1 ng/mL induced about 70% cell death in L929 cells. As shown,the TNF-α-IL6-IL6, chimeric anti-TNF-α clone 6A9 and Ck-AD2-cTNF-α-IgGclone 4A5 were able to neutralize the activity of rhTNF-α, and inhibitcell death in a dose-response manner (FIG. 15), just like their parentantibody 4C9.

TNF-α-(IL6)-(IL6) and Ck-AD2-cTNF-α were also able to neutralize celldeath of L929 cells induced by natural human TNF-α (released from RAPBMCs) (FIG. 16). Upon stimulation by type II collagen for 5 days,natural human TNF-α is released from the cultured PBMCs isolated from arheumatoid arthritis patient (S22). In the presence of 2 μg/mL ofactinomycin-D, TNF-α at 0.1 ng/mL induced about 76% cell death. As shownin FIG. 16, TNF-α-IL6-IL6 and Ck-AD2-cTNF-α-IgG clone 4A5 were able toneutralize the activity of the natural human TNF-α, and inhibit celldeath in a dose-response manner.

The ability of cT*-(c6)-(c6) to bind to IL-6 or TNF-α from rat, monkeyor human was determined by ELISA. The results are summarized in FIG. 17,which shows that the affinity of cT*-(c6)-(c6) for IL-6 or TNF-α fromdifferent species was approximately the same as the individualantibodies, and that the antibodies showed approximately similardissociation constants for human, Cynomolgus monkey and canine antigens.

As can be seen in FIG. 18, STAT3 plays a central role in both TNF-α andIL-6 mediated pathways and disease processes and inhibition of STAT3phosphorylation induced by TNF-α or IL-6 is a reasonable surrogate todetermine the efficacy of anti-TNF-α or anti-IL-6 antibody complexes asmoderators of such disease processes. Because TNF-α and IL-6 playpathogenic roles in the development of a variety of autoimmune, immunedysfunction or inflammatory diseases, including but not limited tosystemic lupus erythematosus, rheumatoid arthritis, inflammatory boweldisease, type II diabetes, obesity, atherosclerosis and cachexia relatedto cancer, the presence results show that bispecificanti-IL-6/anti-TNF-α antibodies, such as cT*-(c6)-(c6), are of use fortreatment of such TNF-α/IL-6 mediated diseases or conditions.

What is claimed is:
 1. A murine, chimeric, humanized or human anti-IL-6antibody or antigen-binding fragment thereof comprising the heavy chainCDR sequences CDR1 (GFTFSRFGMH, SEQ ID NO:107), CDR2 (YIGRGSSTIYYADTVKG,SEQ ID NO:108) and CDR3 (SNWDGAMDY, SEQ ID NO:109) and the light chainCDR sequences CDR1 (RASGNIHNFLA, SEQ ID NO:110), CDR2 (NAETLAD, SEQ IDNO:111) and CDR3 (QHFWSTPWT, SEQ ID NO:112).
 2. The anti-IL-6 antibodyor fragment thereof of claim 1, wherein the antibody allotype isselected from the group consisting of nG1m1, G1m3, nG1m1,2 and Km3. 3.The anti-IL-6 antibody or fragment thereof of claim 1, wherein theantibody or fragment is a naked antibody or fragment.